Method of improving efficacy of biological response-modifying proteins and the exemplary muteins

ABSTRACT

Disclosed is a protein variant which substitutes valine for phenylalanine residue in a binding domain having a biological response-modifying function by binding to a receptor, ligand or substrate. Also, the present invention discloses a DNA encoding the protein variant, a recombinant expression vector to which the DNA is operably linked, a host cell transformed or transfected with the recombinant expression vector, and a method of preparing the protein variant comprising cultivating the host cell and isolating the protein variant from the resulting culture. Further, the present invention discloses a pharmaceutical composition comprising the protein variant and a pharmaceutically acceptable carrier.

This application is a Divisional application of U.S. application Ser.No. 12/499,710, filed on Jul. 8, 2009. U.S. Ser. No. 12/499,710 is aDivisional application of U.S. application Ser. No. 10/519,390, filed onJul. 29, 2005, now U.S. Pat. No. 7,569,361. U.S. Ser. No. 10/519,390 isa national stage application under 35 U.S.C. 371 of PCT Application No.PCT/KR2004/001246 having an international filing date of May 27, 2004.PCT/KR2004/001246 claimed the benefit of Korean Application No.10-2003-0051846, filed Jul. 26, 2003. The entire disclosure of eachforegoing applications is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a protein variant which substitutesvaline for phenylalanine residue in a binding domain having a biologicalresponse-modifying function by binding to a receptor, ligand orsubstrate. More particularly, the present invention relates to a proteinvariant which substitutes valine for phenylalanine residue in an α-helixdomain participating in the binding of a human cytokine protein to acorresponding receptor.

BACKGROUND ART

Many human diseases are caused by the loss of protein function due todefects or an insufficient amount of a protein. To treat such diseases,related proteins have been directly administered to patients. However,many physiologically active proteins used as medicines are easilydegraded in serum before they arrive at target tissues and act therein.For this reason, most physiologically active proteins having therapeuticvalue are excessively or frequently administered to patients to maintainan appropriate concentration capable of offering satisfactorytherapeutic effects.

An approach to solve the above problems is to conjugate withpolyethylene glycol (PEGylation) or microencapsulate physiologicallyactive proteins. However, these methods are cumbersome because targetproteins are primarily produced in microorganisms and purified, and arethen PEGlyated or microencapsulated. In addition, cross-linking mayoccur at undesired positions, which may negatively affect thehomogeneity of final products.

Another approach involves glycosylation. Cell surface proteins andsecretory proteins produced by eukaryotic cells are modified by aglycosylation process. Glycosylation is known to influence in vivostability and function of proteins, as well as their physiologicalproperties. However, since glycosylated proteins can be produced only byeukaryotic cells capable of performing glycosylation, their productionprocess is complicated, and it is difficult to attain homogeneous finalproducts which are glycosylated at all desired positions.

In addition, the conventional techniques all improve the problemsassociated with administration frequency, but do not increase thephysiological efficacy of proteins, leading to excessive dosage. Forexample, NESP developed by the Amgen Company (see U.S. Pat. No.6,586,398) improves the frequent administration by extending thehalf-lives of proteins in the blood, but does not increase the efficacyof proteins, leading to excessive dosage that may induce the productionof blocking antibodies.

An approach used to improve the efficacy of physiologically activeproteins is to mutagenize some amino acid residues of a wild-typeprotein to improve biological activity of the protein. Related proteinvariants are disclosed in the following patent publications: (1) U.S.Pat. No. 5,457,089: human erythropoietin (EPO) variants where thecarboxyl terminal region was altered to increase binding affinity of EPOto its receptor, (2) International Pat. Publication No. 02/077034: humangranulocyte colony stimulating factor (G-CSF) variants where a T-cellepitope was altered to reduce immunogenicity of human G-CSF in humans;(3) International Pat. Publication No. 99/57147: human thrombopoietin(TPO) variants prepared by substituting glutaminic acid at the 115position with lysine, arginine or tyrosine in a TOP protein having anamino acid sequence corresponding to 7th to 151st amino acid residues ofhuman mature TPO; and (4) U.S. Part. Nos. 6,136,563 and 6,022,711 thatdisclose human growth hormone variants having alanine substitutions atthe 18, 22, 25, 26, 29, 65, 168 and 174 positions.

However, the aforementioned protein variants are altered forms made forimproving only therapeutic efficacy regardless of changes in in vivoantigenicity. Thus, the scale, degree and position of these alterationshave high potential to induce immune responses in humans. Antigenicityin humans may cause serious adverse effects (Casadevall et al. N. Eng.J. Med. 2002, vol. 346, p. 469).

DISCLOSURE OF THE INVENTION

It is therefore an object of the present invention to provide biologicalresponse-modifying protein variants having improved pharmacologicalaction, which are capable of maximizing biological response modifyingeffects upon administration and preventing the formation of blockingantibodies through an improvement in efficacy of conventional biologicalresponse-modifying proteins, and methods of preparing such variants.

In one aspect, the present invention provides a protein variant whichsubstitutes valine for phenylalanine residue in a binding domain of aprotein having a biological response-modifying function by binding to areceptor, ligand or substrate.

In another aspect, the present invention provides a DNA encoding aprotein variant which substitutes valine for phenylalanine residue in abinding domain of a protein having a biological response-modifyingfunction by binding to a receptor, ligand or substrate.

In a further aspect, the present invention provides a recombinantexpression vector to which a DNA encoding a protein variant whichsubstitutes valine for phenylalanine residue in a binding domain of aprotein having a biological response-modifying function by binding to areceptor, ligand or substrate is operably linked.

In yet another aspect, the present invention provides a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding a protein variant which substitutes valine forphenylalanine residue in a binding domain of a protein having abiological response-modifying function by binding to a receptor, ligandor substrate is operably linked.

In still another aspect, the present invention provides a method ofpreparing a protein variant, comprising cultivating a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding a protein variant which substitutes valine forphenylalanine residue in a binding domain of a protein having abiological response-modifying function by binding to a receptor, ligandor substrate is operably linked, and isolating the protein variant froma resulting culture.

In still another aspect, the present invention provides a pharmaceuticalcomposition comprising a protein variant which substitutes valine forphenylalanine residue in a binding domain of a protein having abiological response-modifying function by binding to a receptor, ligandor substrate, and a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects, features and other advantages of thepresent invention will be more clearly understood from the followingdetailed description taken in conjunction with the accompanyingdrawings, in which:

FIG. 1A is a multiple alignment of amino acid sequences (SEQ ID NO.: 66to SEQ ID NO: 86) of domains participating in the binding of 4-helixbundle cytokines to corresponding receptors;

FIG. 1B is a multiple alignment of amino acid sequences (SEQ ID NO.: 87to SEQ ID NO: 93) of domains participating in the binding of interferonsto corresponding receptors;

FIG. 2A shows the results of Western blotting of TPO variants accordingto the present invention, (from the leftmost lane: marker; wild-typeTPO; TPO-[F46V]; PO-[F128V]; TPO-[F131V]; and TPO-[F141V];

FIG. 2B shows the results of Western blotting of EPO variants accordingto the present invention, (from the leftmost lane: marker; wild-typeEPO; EPO-[F48V]; EPO-[F138V]; EPO-[F142V]; and EPO-[F148V]);

FIG. 2C shows the results of Western blotting of G-CSF variantsaccording to the present invention, (from the leftmost lane: marker,wild-type G-CSF; G-CSF-[F13V]; G-CSF-[F83V]; G-CSF-[F113V];G-CSF-[F140V]; G-CSF-[F144V]; and G-CSF-[F160V]);

FIG. 3A is a graph showing the relative expression levels of TPOvariants according to the present invention, compared to a wild-typeTPO;

FIG. 3B is a graph showing the relative expression levels of EPOvariants according to the present invention, compared to a wild-typeEPO;

FIG. 3C is a graph showing the relative expression levels of G-CSFvariants according to the present invention, compared to a wild-typeG-CSF;

FIG. 4A shows the results of an ELISA assay for binding affinity of TPOvariants according to the present invention to TPO receptors;

FIG. 4B shows the results of an ELISA assay for binding affinity of EPOvariants according to the present invention to EPO receptors;

FIG. 4C shows the results of an ELISA assay for binding affinity ofG-CSF variants according to the present invention to G-CSF receptors;

FIG. 4D shows the results of an ELISA assay for binding affinity of GHvariants according to the present invention to GH receptors;

FIG. 5A shows the results of an SPR assay for binding affinity of TPOvariants according to the present invention to TPO receptors;

FIG. 5B shows the results of an SPR assay for binding affinity of EPOvariants according to the present invention to EPO receptors;

FIG. 6A shows the results of a FACS analysis for binding affinity of aTPO variant according to the present invention to TPO receptors;

FIG. 6B shows the results of a FACS analysis for binding affinity of anEPO variant according to the present invention to EPO receptors;

FIG. 7A is a graph showing the proliferation rates of TF-1/c-Mpl cellsaccording to the concentration of TPO variants according to the presentinvention;

FIG. 7B is a graph showing the proliferation rates of TF-1 cellsaccording to the concentration of EPO variants according to the presentinvention;

FIG. 7C is a graph showing the proliferation rates of HL60 cellsaccording to the concentration of G-CSF variants according to thepresent invention;

FIG. 7D is a graph showing the proliferation rates of Nb2 cellsaccording to the concentration of GH variants according to the presentinvention;

FIG. 8A is a graph showing the results of a pharmacokinetic assay of aTPO variant according to the present invention, in which the TPO variantwas intravenously injected into rabbits, and serum levels of the TPOvariant were measured;

FIG. 8B is a graph showing the results of a pharmacokinetic assay of anEPO variant according to the present invention, in which the EPO variantwas intravenously injected into rabbits, and serum levels of the EPOvariant were measured;

FIG. 8C is a graph showing the results of a pharmacokinetic assay of anEPO variant according to the present invention, in which the EPO variantwas intraperitoneally injected into mice, and serum levels of the EPOvariant were measured;

FIGS. 9A, 9B and 9C are graphs showing the proliferation rates oferythrocytes, proliferation rates of reticulocytes, and changes inhematocrit, respectively, as results of tests to evaluate in vivoactivity of EPO variants according to the present invention, in miceintraperitoneally injected with the EPO variants; and

FIGS. 10A, 10B and 10C are graphs showing proliferation rates ofplatelets, leukocytes and neutrophils respectively, as results of teststo evaluate the in vivo activity of TPO variants according to thepresent invention, in rats intraperitoneally injected with the TPOvariants.

BEST MODE FOR CARRYING OUT THE INVENTION

Single capital letters standing for amino acids, as used herein,represent the following amino acids according to the standardabbreviations defined by the International Union of Biochemistry

A: Alanine; B: Asparagine or Aspartic acid;

C: Cysteine; D: Aspartic acid; E: Glutamic acid;

F: Phenylalanine; G: Glycine; H: Histidine;

I: Isoleucine; K: Lysine; L: Leucine;

M: Methionine; N: Asparagine; P: Proline;

Q: Glutamine; R.; Arginine; S: Serine;

T: Threonine; V: Valine; W: Tryptophan;

Y: Tyrosine; and Z: Glutamine or Glutamic acid.

The designation “(one capital for an amino acid)(amino acidposition)(one capital for another amino acid)”, as used herein, meansthat the former amino acid is substituted by the latter amino acid atthe designated amino acid position of a certain protein. For example,F48V indicates that the phenylalanine residue at the 48th position of acertain protein is substituted by valine. The amino acid position isnumbered from the N terminus of a mature wild-type protein.

The term “protein variant”, as used herein, refers to a protein that hasan amino acid sequence different from a wild-type form by a substitutionof valine for phenylalanine residue in a protein having physiologicalfunction by binding to a receptor, ligand or substrate, in particular,in a domain participating in the binding to a receptor, ligand orsubstrate. In the present invention, a protein variant is designated forconvenience as “protein name-[(one capital for an amino acid)(amino acidposition)(one capital for another amino acid)]”. For example,TPO-[F131V] indicates a TPO variant in which the phenylalanine residueat position 131 of wild-type TPO is substituted by valine.

The term “biological response-modifying proteins”, as used herein,refers to proteins involved in maintaining homeostasis in the body byinducing the initiation or stop of various biological responsesoccurring in the multicellular body and regulating the responses to beorganically connected to each other. These proteins typically act bybinding to receptors, ligands or substrates.

Proteins capable of being altered according to the present inventioninclude all proteins that have innate function to modulate biologicalresponses by binding receptors, ligands or substrates. Non-limitingexamples of the proteins include cytokines, cytokine receptors, adhesionmolecules, tumor necrosis factor (TNF) receptors, enzymes, receptortyrosine kinases, chemokine receptors, other cell surface proteins, andsoluble ligands. Non-limiting examples of the cytokines include CNTF(cytoneurotrophic factor), GH (growth hormone), IL-1, IL-1Ra(interleukin-1 receptor antagonist), placental lactogen (PL),cardioliphin, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-17,TNF, TGF (transforming growth factor), IFN (interferon), GM-CSF(granulocyte-monocyte colony stimulating factor), G-CSF (granulocytecolony stimulating factor), EPO (erythropoietin), TPO (thrombopoietin),M-CSF (monocyte colony stimulating factor), LIF (leukemia inhibitoryfactor), OSM (oncostatin-M), SCF (stem cell factor), HGF (hepatocytegrowth factor), FGF (fibroblast growth factor), IGF (insulin-like growthfactor), and LPT (Leptin). Non-limiting examples of the cytokinereceptors include growth hormone receptor (GHR), IL-13R, IL-1R, IL-2R,IL-3R, IL-4R, IL-5R, IL-6R, IL-7R, IL-9R, IL-15R, TNFR, TGFR, IFNR(e.g., IFN-γR α-chain, IFN-γR β-chain), interferon-αR, -βR and -γR,GM-CSFR, G-CSFR, EPOR, cMpl, gp130, and Fas (Apo 1). Examples of thechemokine receptors include CCR1 and CXCR1-4. Examples of the receptortyrosine kinases include TrkA, TrkB, TrkC, Hrk, REK7, Rse/Tyro-3,hepatocyte growth factor R, platelet-derived growth factor R, and Flt-1.Examples of other cell surface proteins include CD2, CD4, CD5, CD6,CD22, CD27, CD28, CD30, CD31, CD40, CD44, CD100, CD137, CD150, LAG-3,B7, B61, β-neurexin, CTLA-4, ICOS, ICAM-1, complement R-2(CD21), IgER,lysosomal membrane gp-1, α2-microglobulin receptor-related protein, andnatriuretic peptide receptor.

To improve the efficacy of modulating biological responses for theaforementioned numerous proteins having biological response-modulatingfunction, the present invention intends to provide protein variantscapable of binding to receptors, ligands or substrates having a higherhydrophobic force than that of wild types. For this purpose, the presentinvention is characterized by substituting valine for phenylalanineresidue in a binding domain of each of the proteins.

Phenylalanine is a relatively non-polar amino acid that has an aromaticside chain and a known hydrophobicity index of 3.0. Valine is anon-polar hydrophobic amino acid that has an aliphatic side chain and aknown hydrophobicity index of 4.0. In addition, since valine is smallerthan phenylalanine, a protein substituting valine for phenylalanineresidue becomes more deeply depressed in a pocket binding to acorresponding receptor, ligand or substrate. Thus, a proteinsubstituting valine for phenylalanine residue in a binding domain hasincreased hydrophobic force and a more deeply depressed space so that ithas increased binding affinity to a receptor, ligand or substrate,leading to a desired increase in biological response-modulatingefficiency.

In addition, the valine substitution for phenylalanine residue, as aconservative substitution, has a minimal influence on the secondary ortertiary structure of a protein, and thus rarely affects the function ofthe protein (Argos, EMBO J. 1989, vol. 8, pp. 779-85). Further, becausephenylalanine is mainly present in a highly hydrophobic region, it israrely exposed to the exterior. When such phenylalanine residue issubstituted by valine, a protein becomes more deeply depressed from thesurface due to the higher hydrophobicity of valine. Thus, thissubstitution has a lower potential to induce antibody production. Acertain protein should primarily bind a corresponding receptor, ligandor substrate to modulate a specific biological response. In the casethat the stronger this binding is, the efficacy of modulating abiological response is improved, related proteins all may be alteredaccording to the present invention, and the present invention includesall of the resulting protein variants.

The fact that such a substitution of valine for phenylalanine residueleads to increased binding affinity is supported by the finding of amutation of FcγRIIIa(CD16) expressed on NK cells in human autoimmunediseases. The human receptor protein has a genetic polymorphism. Thatis, individuals are divided into two groups: at position 176 in a regionparticipating in recognizing Fc of an antibody ligand, one group hasphenylalanine, and the other group has valine. Individuals havingphenylalanine at position 176 of the receptor have weakened bindingaffinity to the Fc region of the antibody ligand and are highlysusceptible to systemic lupus erythematosus (SLE) (Jianming Wu et al. J.Clin. Invest. 1997, vol. 100, pp. 1059-70).

On the other hand, as noted above, the present invention ischaracterized by substituting valine for phenylalanine residue in abinding domain of a biological response-modulating protein. The term“binding domain”, as used herein, refers to a portion (that is, domain)of a protein performing its biological function by binding to areceptor, ligand or substrate, and has relatively high hydrophobicityand low antigenicity compared to other regions of the protein. Bindingdomains of proteins are well known in the art. For example, some 4-αhelix bundle cytokines and interferons, which are used in an embodimentof the present invention, are known to have a D-α, helix structure andan A-α helix structure, respectively, that serve as binding domains forcorresponding receptors.

However, a binding domain altered according to the present invention isnot limited to binding domains known in the art. This is because thebinding of a biological response-modulating protein to a receptor,ligand or substrate is influenced by, in addition to amino acid residuesinvolved in direct binding, other several amino acid residues. A“binding domain” of a biological response-modulating protein, alteredaccording to the present invention, further includes about 50 amino acidresidues, preferably about 25 amino acid residues, and more preferablyabout 10 amino acid residues, from both ends of a binding domain knownin the art.

One aspect of the present invention involves cytokines that typicallycontain several α helix structures. Among them, the first and lasthelices from the N-terminus are known as binding domains participatingin binding of cytokines to corresponding cytokine receptors (see FIG.1). α helices responsible for binding of cytokines to correspondingreceptors differ according to the type of cytokines, and are well knownin the art. For example, in IL-2, the second and fifth helices bind tothe p55α receptor among IL-2 receptors, the first helix binds to thep75γ receptor among IL-2 receptors, and the sixth helix binds to gammareceptor (Fernando Bazan, Science J. 1992, vol. 257, pp. 410-2). Asdescribed above, cytokines each have particular helices participating inbinding, but the helices have highly conserved amino acid sequences. Thepresent invention provides a cytokine variant that is capable of bindingto a cytokine receptor with higher affinity than a wild-type cytokine bysubstituting valine for phenylalanine residue in an alpha helixcorresponding to a binding domain of a cytokine.

One aspect related to the cytokines involves the 4-helix bundle familyof cytokines. Such cytokines include CNTF, EPO, Flt3L, GM-CSF, IL-2,IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT, LIF, M-CSF, OSM, PL, SCF, TPOG-CSF, GHR and IFN. These cytokines all have four alpha helices, whichare designated as A-alpha helix, B-alpha helix, C-alpha helix andD-alpha helix, respectively. The D- and A-alpha helices mainlyparticipate in binding to receptors (Fernando Bazan, Immunology today,1990, vol. 11 pp. 350-4, The Cytokine Facts Book, 1994, pp. 104-247).

Among the aforementioned 4-helix bundle cytokines, CNTF, EPO, Flt3L,GM-CSF, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT, LIF, M-CSF, OSM,PL, SCF, TPO, G-CSF and GHR have binding domains which each include aD-alpha helix and a region linking a C-alpha helix and the D-alphahelix. More particularly, the binding domains include amino acidresidues between positions 110 and 180 among amino acid residues of the4-helix bundle cytokines. Therefore, in an aspect, the present inventionprovides a 4-helix bundle cytokine variant that is capable of binding toa corresponding receptor with higher affinity than a wild type bysubstituting valine for phenylalanine among amino acid residues betweenpositions 110 and 180 of a 4-helix bundle cytokine.

Of the aforementioned 4-helix bundle cytokines, interferon (e.g.,IFN-α2A, IFN-α2B, IFN-β, IFN-γ, IFN-ω, IFN-τ) have a binding domain thatcontains an “A-alpha helix”. More particularly, the binding domain ofinterferon includes amino acid residues between positions 1 and 50.Therefore, in another aspect, the present invention provides aninterferon variant that is capable of binding to an interferon receptorhaving higher affinity than a wild type by substituting valine forphenylalanine among amino acid residues between positions 1 and 50 of aninterferon.

On the other hand, the binding domain altered according to the presentinvention may include two or more phenylalanine residues. The two ormore phenylalanine residues may all be substituted by valine. However,because this case leads to a great reduction in protein expressionlevels, preferably only one phenylalanine residue is substituted byvaline. In this regard, the present inventors found that, whenphenylalanine residue present in a highly hydrophobic region issubstituted by valine, the biological response-modulating protein hasmuch improved efficacy. Therefore, in the present invention, thephenylalanine residue to be substituted by valine is preferably selectedin a highly hydrophobic region present in the binding domain specifiedaccording to the present invention. Hydrophobicity for a specific regionof an amino acid sequence comprising a protein may be determined by amethod known in the art (Kyte, J. et al. J. Mol. Biol. 1982, vol. 157,pp. 105-132, Hopp, T. P. et al. Proc. Nat. Mad. Sci. USA, 1981, vol.78(6), pp. 3824-3828).

The variant of a biological response-modulating protein according to thepresent invention may be prepared by chemical synthetic methodsgenerally known in the art (Creighton, Proteins: Structures andMolecular Principles, W.H. Freeman and Co., NY 1983). Representativemethods, but are not limited to, include liquid or solid phasesynthesis, fragment condensation, and F-MOC or T-BOC chemical synthesis(Chemical Approaches to the Synthesis of Peptides and Proteins, Williamset al., Eds., CRC Press, Boca Raton Fla., 1997; A Practical Approach,Atherton & Sheppard, Eds., IR, Press, Oxford, England, 1989).

Alternatively, the protein variant according to the present inventionmay be prepared by recombinant DNA techniques. These techniques includea process of preparing a DNA sequence encoding the protein variantaccording to the present invention. Such a DNA sequence may be preparedby altering a DNA sequence encoding a wild-type protein. In brief, aftera DNA sequence encoding a wild-type protein is synthesized, a codon forphenylalanine is changed to another codon for valine by site-directedmutagenesis, thus generating a desired DNA sequence.

Also, the preparation of a DNA sequence encoding the protein variantaccording to the present invention may be achieved by a chemical method.For example, a DNA sequence encoding the protein variant may besynthesized by a chemical method using an oligonucleotide synthesizer.An oligonucleotide is made based on an amino acid sequence of a desiredprotein variant, and preferably by selecting a appropriate codon used bya host cell producing a protein variant. The degeneracy in the geneticcode, which means that one amino acid is specified by more than onecodon, is well known in the art. Thus, there is a plurality of DNAsequences with degeneracy encoding a specific protein variant, and theyall fall into the scope of the present invention.

A DNA sequence encoding the protein variant according to the presentinvention may or may not include a DNA sequence encoding a signalsequence. The signal sequence, if present, should be recognized by ahost cell selected for the expression of the protein variant. The signalsequence may have a prokaryotic or eukaryotic origin or a combinationalorigin, and may be a signal sequence of a native protein. The employmentof a signal sequence may be determined according to the effect ofexpression of a protein variant as a secretory form in a recombinantcell producing the protein variant. If a selected cell is a prokaryoticcell, a DNA sequence typically does not encode a signal sequence butinstead contains preferably an N-terminal methionine for directexpression of a desired protein, and most preferably, a signal sequencederived from a wild type protein is used.

Such a DNA sequence as prepared above is operably linked to another DNAsequence encoding the protein variant of the present invention, and isinserted into a vector including one or more expression controlsequences regulating the expression of the resulting DNA sequence. Then,a host is transformed or transfected with the resulting recombinantexpression vector. The resulting transformant or transfectant iscultured in a suitable medium under suitable conditions for theexpression of the DNA sequence. A substantially pure variant of abiological response-modulating protein coded by the DNA sequence isrecovered from the resulting culture.

The term “vector”, as used herein, means a DNA molecule serving as avehicle capable of stably carrying exogeneous genes into host cells. Foruseful application, a vector should be replicable, have a system forintroducing itself into a host cell, and possess selectable markers. Inaddition, the term “recombinant expression vector”, as used herein,refers to a circular DNA molecule carrying exogeneous genes operablylinked thereto to be expressed in a host cell. When introduced into ahost cell, the recombinant expression plasmid has the ability toreplicate regardless of host chromosomal DNA at a high copy number andto produce heterogeneous DNA. As generally known in the art, in order toincrease the expression level of a transfected gene in a host cell, thegene should be operably linked to transcription and translationregulatory sequences functional in a host cell selected as an expressionsystem. Preferably, the expression regulation sequences and theexogeneous genes may be carried in a single expression vector containingbacteria-selectable markers and a replication origin. In the case thateukaryotic cells are used as an expression system, the expression vectorshould further comprise expression markers useful in the eukaryotic hostcells.

The term “expression control sequences”, as used herein in connectionwith a recombinant expression vector, refers to nucleotide sequencesnecessary or advantageous for expression of the protein variantaccording to the present invention. Each control sequence may be nativeor foreign to the nucleotide sequence encoding the protein variant.Non-limiting examples of the expression control sequences include leadersequences, polyadenylation sequences, propeptide sequences, promoters,enhancers or upstream activating sequences, signal peptide sequences,and transcription terminators. The expression control sequence containsat least one promoter sequence.

The term “operably linked” refers to a state in which a nucleotidesequence is arranged with another nucleotide sequence in a functionalrelationship. The nucleotide sequences may be a gene and controlsequences, which are linked in such a manner that gene expression isinduced when a suitable molecule (for example, transcription-activatingprotein) binds to the control sequence(s). For example, when apre-sequence or secretory leader facilitates secretion of a matureprotein, it is referred to as “operably linked to the protein”. Apromoter is operably linked with a coding sequence when it regulatestranscription of the coding sequence. A ribosome-binding site isoperably linked to a coding sequence when it is present at a positionallowing translation of the coding sequence. Typically, the term“operably linked” means that linked nucleotide sequences are in contactwith each other. In the case of a secretory leader sequence, the termmeans that it contacts a coding sequence and is present within a leadingframe of the coding sequence. However, an enhancer need not necessarilycontact a coding sequence. Linkage of the nucleotide sequences may beachieved by ligation at convenient restriction enzyme recognition sites.In the absence of restriction enzyme recognition sites, oligonucleotideadaptors or linkers may be used, which are synthesized by theconventional methods.

In order to express a DNA sequence encoding the protein variantaccording to the present invention, a wide variety of combinations ofhost cells and vectors as an expression system may be used. Expressionvectors useful for transforming eukaryotic host cells contain expressionregulation sequences from, for example, SV40, bovine papillomavirus,adenovirus, adeno-associated viruses, cytomegalovirus and retroviruses.Expression vectors useful in bacterial host cells include bacterialplasmids from E. coli, which are exemplified by pET, pRSET, pBluescript,pGEX2T, pUC, pBR322, pMB9 and derivatives thereof plasmids having abroad range of host cells, such as RP4, phage DNAs, exemplified by awide variety of λ phage derivatives including λ gt10, λ gt11 and NM989,and other DNA phages, exemplified by filamentous single-stranded DNAphages such as M13. Expression vectors useful in yeast cells include 2μplasmid and derivatives thereof. Expression vectors useful in insectcells include pVL941.

To express a DNA sequence encoding the protein variant according to thepresent invention, any of a wide variety of expression control sequencesmay be used by these vectors. Such useful expression control sequencesinclude those associated with structural genes of the aforementionedexpression vectors. Examples of useful expression control sequencesinclude the early and later promoters of SV40 or adenoviruses, the lacsystem, the trp system, the TAC or TRC system, T3 and T7 promoters, themajor operator and promoter regions of phage λ, the control regions forfd coat protein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of phosphatases, for example, Pho5,the promoters of the yeast alpha-mating system and other sequences knownto control the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof. In particular, T7 RNApolymerase promoter Φ10 is useful for expressing a polypeptide in E.coli.

Host cells transformed or transfected with the aforementionedrecombinant expression vector comprise another aspect of the presentinvention. A wide range of mononuclear host cells may be used forexpressing a DNA sequence encoding the protein variant of the presentinvention. Examples of the host cells include prokaryotic and eukaryoticcells such as E. coli, Pseudomonas sp., Bacillus sp., Streptomyces sp.,fungi or yeasts, insect cells such as Spodoptera frugiperda (Sf9),animal cells such as Chinese hamster ovary cells (CHO) or mouse cells,African green monkey cells such as COS 1, COS 7, BSC 1, BSC 40 or BMT10, and tissue-cultured human and plant cells. Preferred hosts includebacteria such as E. coli and Bacillus subtilis, and tissue-culturedmammalian cells.

The transformation and transfection may be performed by the methodsdescribed in basic experimental guidebooks (Davis et al., Basic Methodsin Molecular Biology, 1986; Sambrook, J., et al., Basic Methods inMolecular Biology, 1989). The preferred methods for introducing a DNAsequence encoding the protein variant according to the present inventioninto a host cell include, for example, calcium phosphate transfection,DEAE-dextran mediated transfection, transvection, microinjection,cationic lipid-mediated transfection, electroporation, transduction,scrape loading, ballistic introduction, and infection.

Also, it will be understood that all vectors and expression controlsequences do not function equally in expressing the DNA sequence of thepresent invention. Likewise, all hosts do not function equally for anidentical expression system. However, those skilled in the art are ableto make a suitable selection from various vectors, expression controlsequences and hosts, within the scope of the present invention, withouta heavy experimental burden. For example, a vector may be selectedtaking a host cell into consideration because the vector should bereplicated in the host cell. The copy number of a vector, ability tocontrol the copy number, and expression of other proteins encoded by thevector, for example, an antibiotic marker, should be deliberated. Also,an expression control sequence may be selected taking several factorsinto consideration. For example, relative strength, control capacity andcompatibility with the DNA sequence of the present invention of thesequence, particularly with respect to possible secondary structures,should be deliberated. Further, the selection of a host cell may be madeunder consideration of compatibility with a selected vector, toxicity ofa product encoded by a nucleotide sequence, secretory nature of theproduct, ability to correctly fold a polypeptide, fermentation orcultivation requirements, ability to ensure easy purification of aproduct encoded by a nucleotide sequence, or the like.

In the method of preparing the protein variant according to the presentinvention, the host cells are cultivated in a nutrient medium suitablefor production of a polypeptide using methods known in the art. Forexample, the cells may be cultivated by shake flask cultivation,small-scale or large-scale fermentation in laboratory or industrialfermenters performed in a suitable medium and under conditions allowingthe polypeptide to be expressed and/or isolated. The cultivation takesplace in a suitable nutrient medium containing carbon and nitrogensources and inorganic salts, using procedures known in the art. Suitablemedia are commercially available from commercial suppliers and may beprepared according to published compositions (for example, the catalogof American Type Culture Collection). If the polypeptide is secretedinto the nutrient medium, the polypeptide can be recovered directly fromthe medium. If the polypeptide is not secreted, it can be recovered fromcell lysates.

The biological response-modulating protein variant according to thepresent invention may be recovered by methods known in the art. Forexample, the protein variant may be recovered from the nutrient mediumby conventional procedures including, but not limited to,centrifugation, filtration, extraction, spray drying, evaporation, orprecipitation. Further, the protein variant may be purified by a varietyof procedures known in the art including, but not limited to,chromatography (e.g., ion exchange, affinity, hydrophobicity, and sizeexclusion), electrophoresis, differential solubility (e.g., ammoniumsulfate precipitation), SDS-PAGE, or extraction.

The present invention provides a pharmaceutical composition comprising avariant of a biological response-modulating protein and apharmaceutically acceptable carrier. In the pharmaceutical compositionaccording to the present invention, the biological response-modulatingprotein variant is preferably contained in a therapeutically effectiveamount.

The carrier used in the pharmaceutical composition of the presentinvention includes the commonly used carriers, adjuvants and vehicles,in the pharmaceutical field, which are as a whole called“pharmaceutically acceptable carriers”. Non-limiting pharmaceuticallyacceptable carriers useful in the pharmaceutical composition of thepresent invention include ion exchange, alumina, aluminum stearate,lecithin, serum proteins (e.g., human serum albumin), buffering agents(e.g., sodium phosphate, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of vegetable saturated fatty acids), water,salts or electrolytes (e.g., protamine sulfate, disodium hydrophosphate,potassium hydrophosphate, sodium chloride, and zinc salts), colloidalsilica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-basedsubstrates, polyethylene glycol, sodium carboxymethylcellulose,polyarylate, waxes, polyethylene-polyoxypropylene-block copolymers,polyethylene glycol, and wool fat.

The pharmaceutical composition of the present invention may beadministered via any of the common routes, if it is able to reach adesired tissue. Therefore, the pharmaceutical composition of the presentinvention may be administered topically, orally, parenterally,intraocularly, transderrnally, intrarectally and intraluminally, and maybe formulated into solutions, suspensions, tablets, pills, capsules andsustained release preparations. The term “parenteral”, as used herein,includes subcutaneous, intranasal, intravenous, intraperitoneal,intramuscular, intra-articular, intra-synovial, intrasternal,intracardial, intrathecal, intralesional and intracranial injection orinfusion techniques.

In an aspect, the pharmaceutical composition of the present inventionmay be formulated as aqueous solutions for parenteral administration.Preferably, a suitable buffer solution, such as Hank's solution,Ringer's solution or physiologically buffered saline, may be employed.Aqueous injection suspensions may be supplemented with substancescapable of increasing viscosity of the suspensions, which areexemplified by sodium carboxymethylcellulose, sorbitol and dextran. Inaddition, suspensions of the active components, such as oily injectionsuspension, include lipophilic solvents or carriers, which areexemplified by fatty oils such as sesame oil, and synthetic fatty acidesters such as ethyl oleate, triglycerides or liposomes. Polycationicnon-lipid amino polymers may also be used as vehicles. Optionally, thesuspensions may contain suitable stabilizers or drugs to increase thesolubility of protein variants and obtain high concentrations of theprotein variants.

The pharmaceutical composition of the present invention is preferably inthe form of a sterile injectable preparation, such as a sterileinjectable aqueous or oleaginous suspension. Such suspension may beformulated according to the methods known in the art, using suitabledispersing or wetting agents (e.g., Tween 80) and suspending agents. Thesterile injectable preparations may also be a sterile injectablesolution or suspension in a non-toxic parenterally-acceptable diluent orsolvent, such as a solution in 1,3-butanediol. The acceptable vehiclesand solvents include mannitol, water, Ringer's solution and isotonicsodium chloride solution. In addition, sterile fixed oils mayconventionally be employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed, including synthetic mono-or di-glycerides. In addition, fatty acids, such as oleic acid andglyceride derivatives thereof may be used in the preparation ofinjectable preparations, like the pharmaceutically acceptable naturaloils (e.g., olive oil or castor oil), and particularly, polyoxyethylatedderivatives thereof.

The aforementioned aqueous composition is sterilized mainly byfiltration using a filter to remove bacteria, mixing with disinfectantsor in combination with radiation. The sterilized composition can behardened, for example, by freeze-drying to obtain a hardened product,and for practical use, the hardened product is dissolved in sterilizedwater or a sterilized diluted solution.

The term “therapeutically effective amount”, as used herein inconnection with the pharmaceutical composition of the present invention,means an amount in which an active component shows an improved ortherapeutic effect toward a disease to which the pharmaceuticalcomposition of the present invention is applied. The therapeuticallyeffective amount of the pharmaceutical composition of the presentinvention may vary according to the patient's age and sex, applicationsites, administration frequency, administration duration, formulationtypes and adjuvant types. Typically, the pharmaceutical composition ofthe present invention is administered in smaller amounts than awild-type protein, for example, 0.01-1000 μg/kg/day, more preferably0.1-500 μg/kg/day, and most preferably 1-100 μg/kg/day.

On the other hand, it will be apparent to those skilled in the art thatdiseases to which the present composition is applied may vary accordingto the protein type. The EPO and TPO altered as in an embodiment of thepresent invention may be used for treating, in addition to anemiaitself, anemia as a complication associated with other diseases (e.g.,anemia in inflammatory bowel disease, Progressive Kidney Disease, anemiaof renal failure, the anemia associated with HIV infection in zidovudine(AZT) treated patients, anemia associated with cancer chemotherapy,Huntington's disease (HD), sickle cell anemia, Late HyporegenerativeAnemia in Neonates with Rh Hemolytic Disease after in utero ExchangeTransfusion). In addition, the G-CSF altered according to the presentinvention may be used for treating neutropenia itself and neutropeniadeveloped after bone marrow transplantation or cancer chemotherapy, theGH variants may be used for treating pituitary dwarfism and paediatricchronic renal failure. However, the present invention is not limited tothese applications.

Hereinafter, the present invention provides interferon variants whicheach substitute valine for specific phenylalanine residue of 4-helixbundle cytokines, in detail, CNTF, EPO, Flt3L, G-CSF, GM-CSF, GH, IL-2,IL-3, IL-4, IL-5, IL-12p35, LPT, LIF, M-CSF, OSM, PL, SCF, TPO, IFN-α2A,IFN-α2B, IFN-β, IFN-γ, IFN-ω and IFN-τ.

In one specific aspect, the present invention provides the followingprotein variants: (1) a CNTF variant that substitutes valine for thephenylalanine residue at the position 3, 83, 98, 105, 119, 152 or 178 ofan amino acid sequence (SEQ ID NO.: 1) of a wild-type CNTF; (2) an EPOvariant that substitutes valine for the phenylalanine residue at theposition 48, 138, 142 or 148 of an amino acid sequence (SEQ ID NO.: 2)of a wild-type EPO; (3) a Flt3L variant that substitutes valine for thephenylalanine residue at the position 6, 15, 81, 87, 96 or 124 of anamino acid sequence (SEQ ID NO.: 3) of a wild-type Flt3L; (4) a G-CSFvariant that substitutes valine for the phenylalanine residue at theposition 13, 83, 113, 140, 144 or 160 of an amino acid sequence (SEQ IDNO.: 4) of a wild-type G-CSF; (5) a GM-CSF variant that substitutesvaline for the phenylalanine residue at the position 47, 103, 106, 113or 119 of an amino acid sequence (SEQ ID NO.: 5) of a wild-type GM-CSF;(6) a GH variant that substitutes valine for the phenylalanine residueat the position 1, 10, 25, 31, 44, 54, 92, 97, 139, 146, 166, 176 or 191of an amino acid sequence (SEQ ID NO.: 6) of a wild-type GH; (7) anIFN-α2A variant that substitutes valine for the phenylalanine residue atthe position 27, 36, 38, 43, 47, 64, 67, 84, 123 or 151 of an amino acidsequence (SEQ ID NO.: 7) of a wild-type IFN-α2A; (8) an IFN-α2B variantthat substitutes valine for the phenylalanine residue at the position27, 36, 38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence(SEQ ID NO.: 8) of a wild-type IFN-α2B; (9) an IFN-β variant thatsubstitutes valine for the phenylalanine residue at the position 8, 38,50, 67, 70, 111 or 154 of an amino acid sequence (SEQ ID NO.: 9) of awild-type IFN-β; (10) an IFN-γ variant that substitutes valine for thephenylalanine residue at the position 18, 32, 55, 57, 60, 63, 84, 85, 95or 139 of an amino acid sequence (SEQ ID NO.: 10) of a wild-type IFN-γ;(11) an IFN-ω variant that substitutes valine for the phenylalanineresidue at the position 27, 36, 38, 65, 68, 124 or 153 of an amino acidsequence (SEQ ID NO.: 11) of a wild-type IFN-ω, (12) an IFN-τ variantthat substitutes valine for the phenylalanine residue at the position 8,39, 68, 71, 88, 127, 156, 157, 159 or 183 of an amino acid sequence (SEQID NO.: 12) of a wild-type IFN-τ; (13) an IL-2 variant that substitutesvaline for the phenylalanine residue at the position 42, 44, 78, 103,117 or 124 of an amino acid sequence (SEQ ID NO.: 13) of a wild-typeIL-2; (14) an IL-3 variant that substitutes valine for the phenylalanineresidue at the position 37, 61, 107, 113 or 133 of an amino acidsequence (SEQ ID NO.: 14) of a wild-type IL-3; (15) an IL-4 variant thatsubstitutes valine for the phenylalanine residue at the position 33, 45,55, 73, 82 or 112 of an amino acid sequence (SEQ ID NO.: 15) of awild-type IL-4; (16) an IL-5 variant that substitutes valine for thephenylalanine residue at the position 49, 69, 96 or 103 of an amino acidsequence (SEQ ID NO.: 16) of a wild-type IL-5; (17) an IL-6 variant thatsubstitutes valine for the phenylalanine residue at the position 73, 77,93, 104, 124, 169 or 172 of an amino acid sequence (SEQ ID NO.: 17) of awild-type IL-6; (18) an IL-12p35 variant that substitutes valine for thephenylalanine residue at the position 13, 39, 82, 96, 116, 132, 150, 166or 180 of an amino acid sequence (SEQ ID NO.: 18) of a wild-typeIL-12p35; (19) a LPT variant that substitutes valine for thephenylalanine residue at the position 41 or 92 of an amino acid sequence(SEQ ID NO.: 19) of a wild-type LPT; (20) a LIF variant that substitutesvaline for the phenylalanine residue at the position 41, 52, 67, 70, 156or 180 of an amino acid sequence (SEQ ID NO.: 20) of a wild-type LIF;(21) a M-CSF variant that substitutes valine for the phenylalanineresidue at the position 35, 37, 54, 67, 91, 106, 121, 135, 143, 229,255, 311, 439, 466 or 485 of an amino acid sequence (SEQ ID NO.: 21) ofa wild-type M-CSF; (22) an OSM variant that substitutes valine for thephenylalanine residue at the position 56, 70, 160, 169, 176 or 184 of anamino acid sequence (SEQ ID NO.: 22) of a wild-type OSM; (23) a PLvariant that substitutes valine for the phenylalanine residue at theposition 10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191 of an aminoacid sequence (SEQ ID NO.: 23) of a wild-, type PL; (24) a SCF variantthat substitutes valine for the phenylalanine residue at the position63, 102, 110, 115, 116, 119, 126, 129, 158, 199, 205, 207 or 245 of anamino acid sequence (SEQ ID NO.: 24) of a wild-type SCF; and (25) a TPOvariant that substitutes valine for the phenylalanine residue at theposition 46, 128, 131, 141, 186, 204, 240 or 286 of an amino acidsequence (SEQ ID NO.: 25) of a wild-type TPO.

In another specific aspect, the present invention provides the followingDNA molecules: (1) a DNA encoding a CNTF variant that substitutes valinefor the phenylalanine residue at the position 3, 83, 98, 105, 119, 152or 178 of an amino acid sequence (SEQ ID NO.: 1) of a wild-type CNTF;(2) a DNA encoding an EPO variant that substitutes valine for thephenylalanine residue at the position 48, 138, 142 or 148 of an aminoacid sequence (SEQ ID NO.: 2) of a wild-type EPO; (3) a DNA encoding aFlt3L variant that substitutes valine for the phenylalanine residue atthe position 6, 15, 81, 87, 96 or 124 of an amino acid sequence (SEQ IDNO.: 3) of a wild-type Flt3L; (4) a DNA encoding a G-CSF variant thatsubstitutes valine for the phenylalanine residue at the position 13, 83,113, 140, 144 or 160 of an amino acid sequence (SEQ ID NO.: 4) of awild-type G-CSF; (5) a DNA encoding a GM-CSF variant that substitutesvaline for the phenylalanine residue at the position 47, 103, 106, 113or 119 of an amino acid sequence (SEQ ID NO.: 5) of a wild-type GM-CSF;(6) a DNA encoding a GH variant that substitutes valine for thephenylalanine residue at the position 1, 10, 25, 31, 44, 54, 92, 97,139, 146, 166, 176 or 191 of an amino acid sequence (SEQ ID NO.: 6) of awild-type GH; (7) a DNA encoding an IFN-α2A variant that substitutesvaline for the phenylalanine residue at the position 27, 36, 38, 43, 47,64, 67, 84, 123 or 151 of an amino acid sequence (SEQ ID NO.: 7) of awild-type IFN-α2A; (8) a DNA encoding an IFN-α2B variant thatsubstitutes valine for the phenylalanine residue at the position 27, 36,38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence (SEQ IDNO.: 8) of a wild-type IFN-α2B; (9) a DNA encoding an IFN-β variant thatsubstitutes valine for the phenylalanine residue at the position 8, 38,50, 67, 70, 111 or 154 of an amino acid sequence (SEQ ID NO.: 9) of awild-type IFN-β; (10) a DNA encoding an IFN-γ variant that substitutesvaline for the phenylalanine residue at the position 18, 32, 55, 57, 60,63, 84, 85, 95 or 139 of an amino acid sequence (SEQ ID NO.: 10) of awild-type IFN-γ; (11) a DNA encoding an IFN-ω variant that substitutesvaline for the phenylalanine residue at the position 27, 36, 38, 65, 68,124 or 153 of an amino acid sequence (SEQ ID NO.: 11) of a wild-typeIFN-ω; (12) a DNA encoding an IFN-τ variant that substitutes valine forthe phenylalanine residue at the position 8, 39, 68, 71, 88, 127, 156,157, 159 or 183 of an amino acid sequence (SEQ ID NO.: 12) of awild-type IFN-τ; (13) a DNA encoding an IL-2 variant that substitutesvaline for the phenylalanine residue at the position 42, 44, 78, 103,117 or 124 of an amino acid sequence (SEQ ID NO.: 13) of a wild-typeIL-2; (14) a DNA encoding an IL-3 variant that substitutes valine forthe phenylalanine residue at the position 37, 61, 107, 113 or 133 of anamino acid sequence (SEQ ID NO.: 14) of a wild-type IL-3; (15) a DNAencoding an IL-4 variant that substitutes valine for the phenylalanineresidue at the position 33, 45, 55, 73, 82 or 112 of an amino acidsequence (SEQ ID NO.: 15) of a wild-type IL-4; (16) a DNA encoding anIL-5 variant that substitutes valine for the phenylalanine residue atthe position 49, 69, 96 or 103 of an amino acid sequence (SEQ ID NO.:16) of a wild-type IL-5; (17) a DNA encoding an IL-6 variant thatsubstitutes valine for the phenylalanine residue at the position 73, 77,93, 104, 124, 169 or 172 of an amino acid sequence (SEQ ID NO.: 17) of awild-type IL-6; (18) a DNA encoding an IL-12p35 variant that substitutesvaline for the phenylalanine residue at the position 13, 39, 82, 96,116, 132, 150, 166 or 180 of an amino acid sequence (SEQ ID NO.: 18) ofa wild-type IL-12p35; (19) a DNA encoding a LPT variant that substitutesvaline for the phenylalanine residue at the position 41 or 92 of anamino acid sequence (SEQ ID NO.: 19) of a wild-type LPT; (20) a DNAencoding a LIF variant that substitutes valine for the phenylalanineresidue at the position 41, 52, 67, 70, 156 or 180 of an amino acidsequence (SEQ ID NO.: 20) of a wild-type LIF; (21) a DNA encoding aM-CSF variant that substitutes valine for the phenylalanine residue atthe position 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255, 311, 439,466 or 485 of an amino acid sequence (SEQ ID NO.: 21) of a wild-typeM-CSF; (22) a DNA encoding an OSM variant that substitutes valine forthe phenylalanine residue at the position 56, 70, 160, 169, 176 or 184of an amino acid sequence (SEQ ID NO.: 22) of a wild-type OSM; (23) aDNA encoding a PL variant that substitutes valine for the phenylalanineresidue at the position 10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191of an amino acid sequence (SEQ ID NO.: 23) of a wild-type PL; (24) a DNAencoding a SCF variant that substitutes valine for the phenylalanineresidue at the position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199,205, 207 or 245 of an amino acid sequence (SEQ ID NO.: 24) of awild-type SCF; and (25) a DNA encoding a TPO variant that substitutesvaline for the phenylalanine residue at the position 46, 128, 131, 141,186, 204, 240 or 286 of an amino acid sequence (SEQ ID NO.: 25) of awild-type TPO.

In a further specific aspect, the present invention provides thefollowing recombinant expression vectors: (1) a recombinant expressionvector to which a DNA encoding a CNTF variant that substitutes valinefor the phenylalanine residue at the position 3, 83, 98, 105, 119, 152or 178 of an amino acid sequence (SEQ ID NO.: 1) of a wild-type CNTF isoperably linked; (2) a recombinant expression vector to which a DNAencoding an EPO variant that substitutes valine for the phenylalanineresidue at the position 48, 138, 142 or 148 of an amino acid sequence(SEQ ID NO.: 2) of a wild-type EPO is operably linked; (3) a recombinantexpression vector to which a DNA encoding a Flt3L variant thatsubstitutes valine for the phenylalanine residue at the position 6, 15,81, 87, 96 or 124 of an amino acid sequence (SEQ ID NO.: 3) of awild-type Flt3L is operably linked; (4) a recombinant expression vectorto which a DNA encoding a G-CSF variant that substitutes valine for thephenylalanine residue at the position 13, 83, 113, 140, 144 or 160 of anamino acid sequence (SEQ ID NO.: 4) of a wild-type G-CSF is operablylinked; (5) a recombinant expression vector to which a DNA encoding aGM-CSF variant that substitutes valine for the phenylalanine residue atthe position 47, 103, 106, 113 or 119 of an amino acid sequence (SEQ IDNO.: 5) of a wild-type GM-CSF is operably linked; (6) a recombinantexpression vector to which a DNA encoding a GH variant that substitutesvaline for the phenylalanine residue at the position 1, 10, 25, 31, 44,54, 92, 97, 139, 146, 166, 176 or 191 of an amino acid sequence (SEQ IDNO.: 6) of a wild-type GH is operably linked; (7) a recombinantexpression vector to which a DNA encoding an IFN-α2A variant thatsubstitutes valine for the phenylalanine residue at the position 27, 36,38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence (SEQ IDNO.: 7) of a wild-type IFN-α2A is operably linked; (8) a recombinantexpression vector to which a DNA encoding an IFN-α2B variant thatsubstitutes valine for the phenylalanine residue at the position 27, 36,38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence (SEQ IDNO.: 8) of a wild-type IFN-α2B is operably linked; (9) a recombinantexpression vector to which a DNA encoding an IFN-β variant thatsubstitutes valine for the phenylalanine residue at the position 8, 38,50, 67, 70, 111 or 154 of an amino acid sequence (SEQ ID NO.: 9) of awild-type IFN-β is operably linked; (10) a recombinant expression vectorto which a DNA encoding an IFN-γ variant that substitutes valine for thephenylalanine residue at the position 18, 32, 55, 57, 60, 63, 84, 85, 95or 139 of an amino acid sequence (SEQ ID NO.: 10) of a wild-type IFN-γis operably linked; (11) a recombinant expression vector to which a DNAencoding an IFN-ω variant that substitutes valine for the phenylalanineresidue at the position 27, 36, 38, 65, 68, 124 or 153 of an amino acidsequence (SEQ ID NO.: 11) of a wild-type IFN-ω is operably linked; (12)a recombinant expression vector to which a DNA encoding an IFN-τ variantthat substitutes valine for the phenylalanine residue at the position 8,39, 68, 71, 88, 127, 156, 157, 159 or 183 of an amino acid sequence (SEQID NO.: 12) of a wild-type IFN-τ is operably linked; (13) a recombinantexpression vector to which a DNA encoding an IL-2 variant thatsubstitutes valine for the phenylalanine residue at the position 42, 44,78, 103, 117 or 124 of an amino acid sequence (SEQ ID NO.: 13) of awild-type IL-2 is operably linked; (14) a recombinant expression vectorto which a DNA encoding an IL-3 variant that substitutes valine for thephenylalanine residue at the position 37, 61, 107, 113 or 133 of anamino acid sequence (SEQ ID NO.: 14) of a wild-type IL-3 is operablylinked; (15) a recombinant expression vector to which a DNA encoding anIL-4 variant that substitutes valine for the phenylalanine residue atthe position 33, 45, 55, 73, 82 or 112 of an amino acid sequence (SEQ IDNO.: 15) of a wild-type IL-4 is operably linked; (16) a recombinantexpression vector to which a DNA encoding an IL-5 variant thatsubstitutes valine for the phenylalanine residue at the position 49, 69,96 or 103 of an amino acid sequence (SEQ ID NO.: 16) of a wild-type IL-5is operably linked; (17) a recombinant expression vector to which a DNAencoding an IL-6 variant that substitutes valine for the phenylalanineresidue at the position 73, 77, 93, 104, 124, 169 or 172 of an aminoacid sequence (SEQ ID NO.: 17) of a wild-type IL-6 is operably linked;(18) a recombinant expression vector to which a DNA encoding an IL-12p35variant that substitutes valine for the phenylalanine residue at theposition 13, 39, 82, 96, 116, 132, 150, 166 or 180 of an amino acidsequence (SEQ ID NO.: 18) of a wild-type IL-12p35 is operably linked;(19) a recombinant expression vector to which a DNA encoding a LPTvariant that substitutes valine for the phenylalanine residue at theposition 41 or 92 of an amino acid sequence (SEQ ID NO.: 19) of awild-type LPT is operably linked; (20) a recombinant expression vectorto which a DNA encoding a LIF variant that substitutes valine for thephenylalanine residue at the position 41, 52, 67, 70, 156 or 180 of anamino acid sequence (SEQ ID NO.: 20) of a wild-type LIF is operablylinked; (21) a recombinant expression vector to which a DNA encoding aM-CSF variant that substitutes valine for the phenylalanine residue atthe position 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255, 311, 439,466 or 485 of an amino acid sequence (SEQ ID NO.: 21) of a wild-typeM-CSF is operably linked; (22) a recombinant expression vector to whicha DNA encoding an OSM variant that substitutes valine for thephenylalanine residue at the position 56, 70, 160, 169, 176 or 184 of anamino acid sequence (SEQ ID NO.: 22) of a wild-type OSM is operablylinked; (23) a recombinant expression vector to which a DNA encoding aPL variant that substitutes valine for the phenylalanine residue at theposition 10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191 of an aminoacid sequence (SEQ ID NO.: 23) of a wild-type PL is operably linked;(24) a recombinant expression vector to which a DNA encoding a SCFvariant that substitutes valine for the phenylalanine residue at theposition 63, 102, 110, 115, 116, 119, 126, 129, 158, 199, 205, 207 or245 of an amino acid sequence (SEQ ID NO.: 24) of a wild-type SCF isoperably linked; and (25) a recombinant expression vector to which a DNAencoding a TPO variant that substitutes valine for the phenylalanineresidue at the position 46, 128, 131, 141, 186, 204, 240 or 286 of anamino acid sequence (SEQ ID NO.: 25) of a wild-type TPO is operablylinked.

In yet another specific aspect, the present invention provides thefollowing host cells: (1) a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding a CNTF variantthat substitutes valine for the phenylalanine residue at the position 3,83, 98, 105, 119, 152 or 178 of an amino acid sequence (SEQ ID NO.: 1)of a wild-type CNTF is operably linked; (2) a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodingan EPO variant that substitutes valine for the phenylalanine residue atthe position 48, 138, 142 or 148 of an amino acid sequence (SEQ ID NO.:2) of a wild-type EPO is operably linked; (3) a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodinga Flt3L variant that substitutes valine for the phenylalanine residue atthe position 6, 15, 81, 87, 96 or 124 of an amino acid sequence (SEQ IDNO.: 3) of a wild-type Flt3L is operably linked; (4) a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding a G-CSF variant that substitutes valine for thephenylalanine residue at the position 13, 83, 113, 140, 144 or 160 of anamino acid sequence (SEQ ID NO.: 4) of a wild-type G-CSF is operablylinked; (5) a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding a GM-CSF variant thatsubstitutes valine for the phenylalanine residue at the position 47,103, 106, 113 or 119 of an amino acid sequence (SEQ ID NO.: 5) of awild-type GM-CSF is operably linked; (6) a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodinga GH variant that substitutes valine for the phenylalanine residue atthe position 1, 10, 25, 31, 44, 54, 92, 97, 139, 146, 166, 176 or 191 ofan amino acid sequence (SEQ ID NO.: 6) of a wild-type GH is operablylinked; (7) a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding an IFN-α2A variant thatsubstitutes valine for the phenylalanine residue at the position 27, 36,38, 43, 47, 64, 67, 84, 123 or 151 of an amino acid sequence (SEQ IDNO.: 7) of a wild-type IFN-α2A is operably linked; (8) a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding an IFN-α2B variant that substitutes valine for thephenylalanine residue at the position 27, 36, 38, 43, 47, 64, 67, 84,123 or 151 of an amino acid sequence (SEQ ID NO.: 8) of a wild-typeIFN-α2B is operably linked; (9) a host cell transformed or transfectedwith a recombinant expression vector to which a DNA encoding an IFN-βvariant that substitutes valine for the phenylalanine residue at theposition 8, 38, 50, 67, 70, 111 or 154 of an amino acid sequence (SEQ IDNO.: 9) of a wild-type IFN-β is operably linked; (10) a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding an IFN-γ variant that substitutes valine for thephenylalanine residue at the position 18, 32, 55, 57, 60, 63, 84, 85, 95or 139 of an amino acid sequence (SEQ ID NO.: 10) of a wild-type IFN-γis operably linked; (11) a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding an IFN-ω variantthat substitutes valine for the phenylalanine residue at the position27, 36, 38, 65, 68, 124 or 153 of an amino acid sequence (SEQ ID NO.:11) of a wild-type IFN-ω is operably linked; (12) a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding an IFN-τ variant that substitutes valine for thephenylalanine residue at the position 8, 39, 68, 71, 88, 127, 156, 157,159 or 183 of an amino acid sequence (SEQ ID NO.: 12) of a wild-typeIFN-τ is operably linked; (13) a host cell transformed or transfectedwith a recombinant expression vector to which a DNA encoding an IL-2variant that substitutes valine for the phenylalanine residue at theposition 42, 44, 78, 103, 117 or 124 of an amino acid sequence (SEQ IDNO.: 13) of a wild-type IL-2 is operably linked; (14) a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding an IL-3 variant that substitutes valine for thephenylalanine residue at the position 37, 61, 107, 113 or 133 of anamino acid sequence (SEQ ID NO.: 14) of a wild-type IL-3 is operablylinked; (15) a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding an IL-4 variant thatsubstitutes valine for the phenylalanine residue at the position 33, 45,55, 73, 82 or 112 of an amino acid sequence (SEQ ID NO.: 15) of awild-type IL-4 is operably linked; (16) a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodingan IL-5 variant that substitutes valine for the phenylalanine residue atthe position 49, 69, 96 or 103 of an amino acid sequence (SEQ ID NO.:16) of a wild-type IL-5 is operably linked; (17) a host cell transformedor transfected with a recombinant expression vector to which a DNAencoding an IL-6 variant that substitutes valine for the phenylalanineresidue at the position 73, 77, 93, 104, 124, 169 or 172 of an aminoacid sequence (SEQ ID NO.: 17) of a wild-type IL-6 is operably linked;(18) a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding an IL-12p35 variant thatsubstitutes valine for the phenylalanine residue at the position 13, 39,82, 96, 116, 132, 150, 166 or 180 of an amino acid sequence (SEQ ID NO.:18) of a wild-type IL-12p35 is operably linked; (19) a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding a LPT variant that substitutes valine for thephenylalanine residue at the position 41 or 92 of an amino acid sequence(SEQ ID NO.: 19) of a wild-type LPT is operably linked; (20) a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding a LIF variant that substitutes valine for thephenylalanine residue at the position 41, 52, 67, 70, 156 or 180 of anamino acid sequence (SEQ ID NO.: 20) of a wild-type LIF is operablylinked; (21) a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding a M-CSF variant thatsubstitutes valine for the phenylalanine residue at the position 35, 37,54, 67, 91, 106, 121, 135, 143, 229, 255, 311, 439, 466 or 485 of anamino acid sequence (SEQ ID NO.: 21) of a wild-type M-CSF is operablylinked; (22) a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding an OSM variant thatsubstitutes valine for the phenylalanine residue at the position 56, 70,160, 169, 176 or 184 of an amino acid sequence (SEQ ID NO.: 22) of awild-type OSM is operably linked; (23) a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodinga PL variant that substitutes valine for the phenylalanine residue atthe position 10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191 of anamino acid sequence (SEQ ID NO.: 23) of a wild-type PL is operablylinked; (24) a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding a SCF variant that substitutesvaline for the phenylalanine residue at the position 63, 102, 110, 115,116, 119, 126, 129, 158, 199, 205, 207 or 245 of an amino acid sequence(SEQ ID NO.: 24) of a wild-type SCF is operably linked; and (25) a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding a TPO variant that substitutes valine for thephenylalanine residue at the position 46, 128, 131, 141, 186, 204, 240or 286 of an amino acid sequence (SEQ ID NO.: 25) of a wild-type TPO isoperably linked.

In still another specific aspect, the present invention provides thefollowing methods of preparing a protein variant: (1) a method ofpreparing a protein variant, comprising cultivating a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding a CNTF variant that substitutes valine for thephenylalanine residue at the position 3, 83, 98, 105, 119, 152 or 178 ofan amino acid sequence (SEQ ID NO.: 1) of a wild-type CNTF is operablylinked, and isolating the protein variant from a resulting culture; (2)a method of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding an EPO variant that substitutes valine for thephenylalanine residue at the position 48, 138, 142 or 148 of an aminoacid sequence (SEQ ID NO.: 2) of a wild-type EPO is operably linked, andisolating the protein variant from a resulting culture; (3) a method ofpreparing a protein variant, comprising cultivating a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding a Flt3L variant that substitutes valine for thephenylalanine residue at the position 6, 15, 81, 87, 96 or 124 of anamino acid sequence (SEQ ID NO.: 3) of a wild-type Flt3L is operablylinked, and isolating the protein variant from a resulting culture; (4)a method of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding a G-CSF variant that substitutes valine for thephenylalanine residue at the position 13, 83, 113, 140, 144 or 160 of anamino acid sequence (SEQ ID NO.: 4) of a wild-type G-CSF is operablylinked, and isolating the protein variant from a resulting culture; (5)a method of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding a GM-CSF variant that substitutes valine for thephenylalanine residue at the position 47, 103, 106, 113 or 119 of anamino acid sequence (SEQ ID NO.: 5) of a wild-type GM-CSF is operablylinked, and isolating the protein variant from a resulting culture; (6)a method of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding a GH variant that substitutes valine for thephenylalanine residue at the position 1, 10, 25, 31, 44, 54, 92, 97,139, 146, 166, 176 or 191 of an amino acid sequence (SEQ ID NO.: 6) of awild-type GH is operably linked, and isolating the protein variant Loina resulting culture; (7) a method of preparing a protein variant,comprising cultivating a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding, an IFN-α2Avariant that substitutes valine for the phenylalanine residue at theposition 27, 36, 38, 43, 47, 64, 67, 84, 123 or 151 of an amino acidsequence (SEQ ID NO.: 7) of a wild-type IFN-α2A is operably linked, andisolating the protein variant from a resulting culture; (8) a method ofpreparing a protein variant, comprising cultivating a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding an IFN-α2B variant that substitutes valine for thephenylalanine residue at the position 27, 36, 38, 43, 47, 64, 67, 84,123 or 151 of an amino acid sequence (SEQ ID NO.: 8) of a wild-typeIFN-α2B is operably linked, and isolating the protein variant from aresulting culture; (9) a method of preparing a protein variant,comprising cultivating a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding an IFN-β variantthat substitutes valine for the phenylalanine residue at the position 8,38, 50, 67, 70, 111 or 154 of an amino acid sequence (SEQ ID NO.: 9) ofa wild-type IFN-β is operably linked, and isolating the protein variantfrom a resulting culture; (10) a method of preparing a protein variant,comprising cultivating a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding an IFN-γ variantthat substitutes valine for the phenylalanine residue at the position18, 32, 55, 57, 60, 63, 84, 85, 95 or 139 of an amino acid sequence (SEQID NO.: 10) of a wild-type IFN-γ is operably linked, and isolating theprotein variant from a resulting culture; (11) a method of preparing aprotein variant, comprising cultivating a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodingan IFN-ω variant that substitutes valine for the phenylalanine residueat the position 27, 36, 38, 65, 68, 124 or 153 of an amino acid sequence(SEQ ID NO.: 11) of a wild-type IFN-ω is operably linked, and isolatingthe protein variant from a resulting culture; (12) a method of preparinga protein variant, comprising cultivating a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodingan IFN-τ variant that substitutes valine for the phenylalanine residueat the position 8, 39, 68, 71, 88, 127, 156, 157, 159 or 183 of an aminoacid sequence (SEQ ID NO.: 12) of a wild-type IFN-τ is operably linked,and isolating the protein variant from a resulting culture; (13) amethod of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding an IL-2 variant that substitutes valine for thephenylalanine residue at the position 42, 44, 78, 103, 117 or 124 of anamino acid sequence (SEQ ID NO.: 13) of a wild-type IL-2 is operablylinked, and isolating the protein variant from a resulting culture; (14)a method of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding an IL-3 variant that substitutes valine for thephenylalanine residue at the position 37, 61, 107, 113 or 133 of anamino acid sequence (SEQ ID NO.: 14) of a wild-type IL-3 is operablylinked, and isolating the protein variant from a resulting culture; (15)a method of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding an IL-4 variant that substitutes valine for thephenylalanine residue at the position 33, 45, 55, 73, 82 or 112 of anamino acid sequence (SEQ ID NO.: 15) of a wild-type IL-4 is operablylinked, and isolating the protein variant from a resulting culture; (16)a method of preparing a protein variant, comprising cultivating a hostcell transformed or transfected with a recombinant expression vector towhich a DNA encoding an IL-5 variant that substitutes valine for thephenylalanine residue at the position 49, 69, 96 or 103 of an amino acidsequence (SEQ ID NO.: 16) of a wild-type IL-5 is operably linked, andisolating the protein variant from a resulting culture; (17) a method ofpreparing a protein variant, comprising cultivating a host celltransformed or transfected with a recombinant expression vector to whicha DNA encoding an IL-6 variant that substitutes valine for thephenylalanine residue at the position 73, 77, 93, 104, 124, 169 or 172of an amino acid sequence (SEQ ID NO.: 17) of a wild-type IL-6 isoperably linked, and isolating the protein variant from a resultingculture; (18) a method of preparing a protein variant, comprisingcultivating a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding an IL-12p35 variant thatsubstitutes valine for the phenylalanine residue at the position 13, 39,82, 96, 116, 132, 150, 166 or 180 of an amino acid sequence (SEQ ID NO.:18) of a wild-type IL-12p35 is operably linked, and isolating theprotein variant from a resulting culture; (19) a method of preparing aprotein variant, comprising cultivating a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodinga LPT variant that substitutes valine for the phenylalanine residue atthe position 41 or 92 of an amino acid sequence (SEQ ID NO.: 19) of awild-type LPT is operably linked, and isolating the protein variant froma resulting culture; (20) a method of preparing a protein variant,comprising cultivating a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding a LIF variant thatsubstitutes valine for the phenylalanine residue at the position 41, 52,67, 70, 156 or 180 of an amino acid sequence (SEQ ID NO.: 20) of awild-type LIF is operably linked, and isolating the protein variant froma resulting culture; (21) a method of preparing a protein variant,comprising cultivating a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding a M-CSF variantthat substitutes valine for the phenylalanine residue at the position35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255, 311, 439, 466 or 485of an amino acid sequence (SEQ ID NO.: 21) of a wild-type M-CSF isoperably linked, and isolating the protein variant from a resultingculture; (22) a method of preparing a protein variant, comprisingcultivating a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding an OSM variant thatsubstitutes valine for the phenylalanine residue at the position 56, 70,160, 169, 176 or 184 of an amino acid sequence (SEQ ID NO.: 22) of awild-type OSM is operably linked, and isolating the protein variant froma resulting culture; (23) a method of preparing a protein variant,comprising cultivating a host cell transformed or transfected with arecombinant expression vector to which a DNA encoding a PL variant thatsubstitutes valine for the phenylalanine residue at the position 10, 31,44, 52, 54, 92, 97, 146, 166, 176 or 191 of an amino acid sequence (SEQID NO.: 23) of a wild-type PL is operably linked, and isolating theprotein variant from a resulting culture; (24) a method of preparing aprotein variant, comprising cultivating a host cell transformed ortransfected with a recombinant expression vector to which a DNA encodinga SCF variant that substitutes valine for the phenylalanine residue atthe position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199, 205, 207or 245 of an amino acid sequence (SEQ ID NO.: 24) of a wild-type SCF isoperably linked, and isolating the protein variant from a resultingculture; and (25) a method of preparing a protein variant, comprisingcultivating a host cell transformed or transfected with a recombinantexpression vector to which a DNA encoding a TPO variant that substitutesvaline for the phenylalanine residue at the position 46, 128, 131, 141,186, 204, 240 or 286 of an amino acid sequence (SEQ ID NO.: 25) of awild-type TPO is operably linked, and isolating the protein variant froma resulting culture.

In still another specific aspect, the present invention provides thefollowing pharmaceutical compositions: (1) a pharmaceutical compositioncomprising a CNTF variant that substitutes valine for the phenylalanineresidue at the position 3, 83, 98, 105, 119, 152 or 178 of an amino acidsequence (SEQ ID NO.: 1) of a wild-type CNTF and a pharmaceuticallyacceptable carrier; (2) a pharmaceutical composition comprising an EPOvariant that substitutes valine for the phenylalanine residue at theposition 48, 138, 142 or 148 of an amino acid sequence (SEQ BD NO.: 2)of a wild-type EPO and a pharmaceutically acceptable carrier, (3) apharmaceutical composition comprising a Flt3L variant that substitutesvaline for the phenylalanine residue at the position 6, 15, 81, 87, 96or 124 of an amino acid sequence (SEQ ID NO.: 3) of a wild-type Flt3Land a pharmaceutically acceptable carrier, (4) a pharmaceuticalcomposition comprising a G-CSF variant that substitutes valine for thephenylalanine residue at the position 13, 83, 113, 140, 144 or 160 of anamino acid sequence (SEQ ID NO.: 4) of a wild-type G-CSF and apharmaceutically acceptable carrier, (5) a pharmaceutical compositioncomprising a GM-CSF variant that substitutes valine for thephenylalanine residue at the position 47, 103, 106, 113 or 119 of anamino acid sequence (SEQ ID NO.: 5) of a wild-type GM-CSF and apharmaceutically acceptable carrier; (6) a pharmaceutical compositioncomprising a GH variant that substitutes valine for the phenylalanineresidue at the position 1, 10, 25, 31, 44, 54, 92, 97, 139, 146, 166,176 or 191 of an amino acid sequence (SEQ ID NO.: 6) of a wild-type GHand a pharmaceutically acceptable carrier; (7) a pharmaceuticalcomposition comprising an IFN-α2A variant that substitutes valine forthe phenylalanine residue at the position 27, 36, 38, 43, 47, 64, 67,84, 123 or 151 of an amino acid sequence (SEQ ID NO.: 7) of a wild-typeIFN-α2A and a pharmaceutically acceptable carrier; (8) a pharmaceuticalcomposition comprising an IFN-α2B variant that substitutes valine forthe phenylalanine residue at the position 27, 36, 38, 43, 47, 64, 67,84, 123 or 151 of an amino acid sequence (SEQ ID NO.: 8) of a wild-typeIFN-α2B and a pharmaceutically acceptable carrier; (9) a pharmaceuticalcomposition comprising an IFN-β variant that substitutes valine for thephenylalanine residue at the position 8, 38, 50, 67, 70, 111 or 154 ofan amino acid sequence (SEQ ID NO.: 9) of a wild-type IFN-β and apharmaceutically acceptable carrier; (10) a pharmaceutical compositioncomprising an IFN-γ variant that substitutes valine for thephenylalanine residue at the position 18, 32, 55, 57, 60, 63, 84, 85, 95or 139 of an amino acid sequence (SEQ ID NO.: 10) of a wild-type IFN-γand a pharmaceutically acceptable carrier; (11) a pharmaceuticalcomposition comprising an IFN-ω variant that substitutes valine for thephenylalanine residue at the position 27, 36, 38, 65, 68, 124 or 153 ofan amino acid sequence (SEQ ID NO.: 11) of a wild-type IFN-ω and apharmaceutically acceptable carrier; (12) a pharmaceutical compositioncomprising an IFN-τ variant that substitutes valine for thephenylalanine residue at the position 8, 39, 68, 71, 88, 127, 156, 157,159 or 183 of an amino acid sequence (SEQ ID NO.: 12) of a wild-typeIFN-τ and a pharmaceutically acceptable carrier; (13) a pharmaceuticalcomposition comprising an IL-2 variant that substitutes valine for thephenylalanine residue at the position 42, 44, 78, 103, 117 or 124 of anamino acid sequence (SEQ ID NO.: 13) of a wild-type IL-2 and apharmaceutically acceptable carrier, (14) a pharmaceutical compositioncomprising an IL-3 variant that substitutes valine for the phenylalanineresidue at the position 37, 61, 107, 113 or 133 of an amino acidsequence (SEQ ID NO.: 14) of a wild-type IL-3 and a pharmaceuticallyacceptable carrier, (15) a pharmaceutical composition comprising an IL-4variant that substitutes valine for the phenylalanine residue at theposition 33, 45, 55, 73, 82 or 112 of an amino acid sequence (SEQ IDNO.: 15) of a wild-type IL-4 and a pharmaceutically acceptable carrier;(16) a pharmaceutical composition comprising an IL-5 variant thatsubstitutes valine for the phenylalanine residue at the position 49, 69,96 or 103 of an amino acid sequence (SEQ ID NO.: 16) of a wild-type IL-5and a pharmaceutically acceptable carrier; (17) a pharmaceuticalcomposition comprising an IL-6 variant that substitutes valine for thephenylalanine residue at the position 73, 77, 93, 104, 124, 169 or 172of an amino acid sequence (SEQ ID NO.: 17) of a wild-type IL-6 and apharmaceutically acceptable carrier; (18) a pharmaceutical compositioncomprising an IL-12p35 variant that substitutes valine for thephenylalanine residue at the position 13, 39, 82, 96, 116, 132, 150, 166or 180 of an amino acid sequence (SEQ ID NO.: 18) of a wild-typeIL-12p35 and a pharmaceutically acceptable carrier; (19) apharmaceutical composition comprising a LPT variant that substitutesvaline for the phenylalanine residue at the position 41 or 92 of anamino acid sequence (SEQ ID NO.: 19) of a wild-type LPT and apharmaceutically acceptable carrier, (20) a pharmaceutical compositioncomprising a LIF variant that substitutes valine for the phenylalanineresidue at the position 41, 52, 67, 70, 156 or 180 of an amino acidsequence (SEQ ID NO.: 20) of a wild-type LIF and a pharmaceuticallyacceptable carrier; (21) a pharmaceutical composition comprising a M-CSFvariant that substitutes valine for the phenylalanine residue at theposition 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255, 311, 439, 466or 485 of an amino acid sequence (SEQ ID NO.: 21) of a wild-type M-CSFand a pharmaceutically acceptable carrier; (22) a pharmaceuticalcomposition comprising an OSM variant that substitutes valine for thephenylalanine residue at the position 56, 70, 160, 169, 176 or 184 of anamino acid sequence (SEQ ID NO.: 22) of a wild-type OSM and apharmaceutically acceptable carrier-, (23) a pharmaceutical compositioncomprising a PL variant that substitutes valine for the phenylalanineresidue at the position 10, 31, 44, 52, 54, 92, 97, 146, 166, 176 or 191of an amino acid sequence (SEQ ID NO.: 23) of a wild-type PL and apharmaceutically acceptable carrier, (24) a pharmaceutical compositioncomprising a SCF variant that substitutes valine for the phenylalanineresidue at the position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199,205, 207 or 245 of an amino acid sequence (SEQ ID NO.: 24) of awild-type SCF and a pharmaceutically acceptable carrier, and (25) apharmaceutical composition comprising a TPO variant that substitutesvaline for the phenylalanine residue at the position 46, 128, 131, 141,186, 204, 240 or 286 of an amino acid sequence (SEQ ID NO.: 25) of awild-type TPO and a pharmaceutically acceptable carrier.

The present purpose to improve the efficacy in modulating biologicalresponses was accomplished in the following examples using TPO, EPO,G-CSF and GH. It will be apparent to those skilled in the art that thefollowing examples are provided only to illustrate the presentinvention, and the scope of the present invention is not limited to theexamples.

Example 1 Construction of DNA Coding Wild Type TPO/EPO/G-CSF/GH

A. Construction of DNA Coding Wild Type TPO

750 μl of TRIzol reagent (MRC., USA) was added to bone marrow tissue ina microcentrifuge tube and incubated at room temperature for 5 minutes.200 μl of chloroform was added into the tube and then the tube wasshaken vigorously for 15 seconds. After incubating the tube at momtemperature for 2-3 minutes, it was centrifuged at 15,000 rpm for 15minutes at 4° C. The upper phase was transferred to a 1.5 μl tube and500 μl of isopropanol was added. The sample was incubated at −70° C. for30 minutes and centrifuged at 15,000 rpm for 15 minutes at 4° C. Afterdiscarding supernatant, RNA pellet was washed once with 75% DEPC-ethanolby vortexing and centrifuged at 15,000 rpm for 15 minutes at 4° C. Thesupernatant was removed and the RNA pellet was dried for 5 minutes atroom temperature and then the pellet was dissolved in 50 μl ofDEPC-treated 3° distilled water.

2 μg of mRNA purified as above and 1 μl of oligo dT30 primer (10 μM,Promega, USA) were mixed and heated at 70° C. for 2 minutes and then itwas immediately cooled on ice for 2 minutes. After that, this reactionmixture was added with 200 U M-MLV reverse transcriptase (Promega, USA),10 μl of 5× reaction buffer (250 mM Tris-HCl, pH 8.3, 375 mM KCl, 15 mMMgCl₂, 50 nM DTT), 1 μl of dNTP (10 mM dATP, 10 mM dTTP, 10 mM dGTP, 10mM dCTP) and DEPC-treated 3° water was added to make the total volume of50 μl. After mixing gently, the reaction mixture was incubated at 42° C.for 60 minutes.

To amplify cDNA coding wild type TPO, the first strand cDNA as template,primer 1 and primer 2 (Table 1) were added into a PCR tube including 2 Uof pfu DNA polymerase (Stratagene, USA), 10 μl of 10× reaction buffer,1% Triton X-100, 1 mg/ml BSA, 3 μl of primer 1 (10 μM), 3 μl of primer 2(10 μM), 2 μl of dNTP (10 mM dATP, 10 mM dTTP, 10 mM dGTP, 10 mM dCTP),and distilled water was added to make the total volume of 100 μl. ThePCR reaction condition was as follows; 1 cycle at 95° C. for 3 minutes,and then 30 cycles at 95° C. for 30 seconds, at 52° C. for 1 minute, andat 72° C. for 1.5 minutes, and finally 1 cycle at 72° C. for 10 minutesto make PCR product with completely blunt end.

The PCR product obtained was separated in 0.8% agarose gel (BMA, USA)and was purified with Qiaex II gel extraction kit (Qiagen, USA). Afterthe isolated DNA was mixed with 15 U of EcoRI, 10 U of NotI, 3 μl of 10×reaction buffer and 3° distilled water was added to make the totalvolume of 30 μl, DNA was restricted by incubation at 37° C. for 2 hours.The PCR product was separated in 0.8% agarose gel and was purified withQiaex II gel extraction kit.

After 5 μg of the pBluescript KS II(+) vector was mixed with 15 U ofEcoRI., 10 U of NotI, 3 μl of 10× reaction buffer and 3° distilled waterwas added to make the total volume of 30 μl, DNA was restricted byincubation at 37° C. for 2 hours. The restricted pBluescript KS II (+)vector was separated in 0.8% agarose gel and was purified with Qiaex IIgel extraction kit.

100 ng of the digested pBluescript KS II(+) vector was ligated with 20ng of the PCR product which was digested with same enzymes. Thisligation mixture was incubated at 16° C. water bath for 16 hours, thusproducing a recombinant vector comprising cDNA coding wild type TPO.Then, it was transformed into a E. coli Top 10 (Invitrogen, USA) whichwas made to a competent cell by rubidium chloride method. Thetransformed bacteria was cultured on LB agar plate containing 50 μg/mlof ampicillin (Sigma, USA). After overnight incubation, colonies weretransferred into tubes with 3 ml of LB medium containing 50 μg/mlampicillin and then they were cultured at 37° C. for 16 hours. Plasmidwas isolated from the cultured bacteria with alkaline lysis method andthe restriction of EcoRI/NotI was used to detect inclusion of clonedgene in the plasmid.

B. Constriction of DNA Coding Wild Type EPO

Procedure of cloning DNA coding wild type EPO was basically same to thatused for cloning DNA coding wild type TPO.

The first strand cDNA as template, primer 11 and primer 12 (Table 2)were used for PCR amplification of DNA coding wild type EPO. The PCRproduct and cloning vector, pBluescript KS II(+) were digested with bothEcoRI and BamHI endonucleases. The digested PCR product and cloningvector were ligated and transformed into competent cell, E. coli Top 10(Invitrogen, USA). Plasmid was isolated from the cultured bacteria withalkaline lysis method and the restriction of EcoRI/BamHI was used todetect existence of cloned gene in the plasmid.

C. Construction of DNA Coding Wild Type G-CSF

Construction procedure of DNA coding wild type G-CSF was similar to thatused for DNA coding wild type TPO.

Leukocytes from healthy people were used for the mRNA extraction, andprimers 21 and 22 (Table 3) were used for PCR amplification of cDNAcoding wild type G-CSF. Both the PCR product and cloning vector,pBluescript KS II(+) were digested with SmaI and EcoRI endonuclease. Thedigested PCR product and cloning vector were ligated and transformedinto competent cell, E. coli Top 10 (Invitrogen, USA). Plasmid wasisolated from the cultured bacteria with alkaline lysis method and therestriction of SmaI/EcoRI was used to detect existence of cloned gene inthe plasmid.

D. Construction of DNA Coding Wild Type GH

DNA coding wild type GH was purchased from ATCC (ATCC No. 67097). To addleader sequence to N-terminal end of this cDNA, primer 35 and 36 (Table4) were used for PCR. In order to make complete cDNA coding wild type GHlinked to the leader sequence, secondary PCR was carried out usingprimers 37 and 38 (Table 4). The PCR product and cloning vector,pBluescript KS II(+) were digested with EcoRI and HindIII endonuclease.Plasmid was isolated from the cultured bacteria with alkaline lysismethod and the restriction of EcoRI/HindIII was used to detect existenceof cloned gene in the plasmid.

Example 2 Construction of cDNA Coding TPO/EPO/G-CSF/GH Muteins

A. Construction of cDNAs Coding TPO Muteins

Four muteins of TPO, TPO-[F46V], TPO-[F128V], TPO-[F131V] andTPO-[F141V] were constructed according to procedures as follows to havea single amino acid-substitution from phenylalanine to valine at eachpositions, respectively.

TABLE 1 Primers used in constructing cDNAs coding TPO- wild type and muteinsPrimer No. Nucleotide sequence Sequence No. 1 Wild type TPO Sense5′-CGGAATTCGGATGGAGCTGACTGAATTG-3′ 26 2 Antisense5′-TTTAGCGGCCGCATTCTTACCCTTCCTGAG-3′ 27 3 TPO-[F46V] Sense T3 4Antisense 5′-CCAAGCTAACGTCCACAGCAG-3′ 28 5 TPO-[F128V] Sense T3 6antisense 5′-GCTCAGGACGATGGCAT-3′ 29 7 TPO-[F131V] Sense T3 8 antisense5′-GGTGTTGGACGCTCAGGAAGATG-3′ 30 9 TPO-[F141V] Sense T3 10 antisense5′-CATCAGGACACGCACCTTTCC-3′ 31

cDNA which code TPO-[F46V], TPO-[F128V], TPO-[F131V] and TPO-[F141V] wasconstructed by primary PCR using specific primers (Table 1) anduniversal primer T3 and secondary PCR using the primary PCR product anduniversal primer T7. The template for these reactions was the cDNAcoding wild type TPO cloned in pBluescript KS II(+) obtained fromExample 1.

The primary PCR was performed by adding 2.5 U Ex taq (Takara, Japan), 5μl of 10× buffer, 1 mM MgCl₂, 2.5 mM dNTP and D.W was added to make thetotal volume of 50 μl. The PCR condition consisted of 1 cycle at 94° for3 minutes followed by 30 cycles at 95° C. for 30 seconds, at 60° C. for30 seconds and at 72° C. for 30 seconds and then linked to 1 cycle at72° C. for 7 minutes. The primary PCR product was used as a megaprimerin the secondary PCR together with universal primer T7 (10 pmole). ThecDNA coding wild type TPO cloned in pBluescript KS II(+) was used as thetemplate in the secondary PCR. The secondary PCR was performed by adding2.5 U Ex taq, 5 μl of 10× buffer, 2.5 mM dNTP and D.W was added to makethe total volume of 50 μl. The PCR condition consisted of 1 cycle at 94°C. for 3 minutes followed by 30 cycles at 94° C. for 1 minute, at 58° C.for 1 minute, and at 72° C. for 1.5 minutes and finally linked to 1cycle at 72° C. for 7 minutes prior to termination.

To minimize errors derived form DNA synthesis, Mg²⁺ concentration wasreduced to 1 mM in the primary PCR. Sizes of megaprimers amplified were280 b.p for TPO-[F46V], 520 b.p for TPO-[F128V], 530 b.p for TPO-[F131V]and 560 b.p for TPO-[F141V]. In the secondary PCR using megaprimers,cDNA coding each muteins produced showed the same size of 1062 b.p.Substitution from phenylalanine to valine at nucleotide sequence of theindividual TPO mutein was verified by direct sequencing.

Each PCR product of 1062 b.p was separated in 0.8% agarose gel andpurified with Qiaex II gel extraction kit. The PCT product was digestedwith 15 U EcoRI and 10 U NotI at 37° C. for 2 hours. The digested PCRproduct was separated in 0.8% agarose gel and purified with Qiaex II gelextraction kit and ligated with pBluescript KS II(+) as described above.The recombinant expression vector containing DNA which codes TPO-[F141V]was named Tefficacin-4 and was deposited at the KCCM (Korean CultureCenter of Microorganisms) under the Budapest Treaty on Jun. 9, 2003.Accession number given by international depositary authority wasKCCM-10500.

B. Construction of cDNAs Coding EPO Muteins

Four muteins of EPO, EPO-[F48V], EPO-[F138V], EPO-[F142VJ] andEPO-[F148V] were constructed according to procedures as follows to havea single amino acid-substitution from phenylalanine to valine at eachpositions, respectively.

TABLE 2Primers used in constructing cDNAs coding EPO- wild type and muteinsPrimer No. Nucleotide sequence Sequence No. 11 Wild EPO Sense5′-GGCGCGGAGATGGGGGT-3′ 32 12 Antisense 5′-TGGTCATCTGTCCCCTGTCCTG-3′ 3313 EPO-[F48V] Sense T3 14 Antisense 5′-GACATTAACTTIGGTGTCTGGGAC-3′ 34 15EPO-[F138V] Sense 5′-CTGTCCCGCAAACTCTTCCGAG-3′ 35 16 Antisense T7 17EPO-[F142V] Sense 5′-CGCAAACTCGTCCGAGTCTACT-3′ 36 18 Antisense T7 19EPO-[F148V] Sense 5′-GAGTCTACTCCAATGTGGTGGG-3′ 37 20 Antisense T7

Construction procedure of cDNA coding EPO muteins was basically similarto that of TPOs. cDNAs which code EPO-[F48V], EPO-[F138V], EPO-[F142V],and EPO-[F148V] were constructed by primary PCR using specific primers(Table 2) and universal primer T3 and secondary PCR using the primaryPCR product and universal primer T7. The template for these reactionswas the cDNA coding wild type EPO cloned in pBluescript KS II(+)obtained from Example 1.

Mg²⁺ concentration was adjusted to 1 mM in the primary PCR. Sizes ofamplified megaprimers were 300 b.p for EPO-[F48V], 550 b.p forEPO-[F138V], 550 b.p for FPO-[F142V] and 550 b.p for EPO-[F148V]. In thesecondary PCR using the megaprimers, cDNAs coding the individual muteinswere amplified as the same size of 580 b.p. Substitution fromphenylalanine to valine at nucleotide sequence of the individual EPOmutein was verified by direct sequencing.

Each PCR product of 580 b.p was separated in 0.8% agarose gel and waspurified with Qiaex II gel extraction kit. The PCR product was digestedwith 15 U EcoRI and 10 U BamHI at 37° C. for 2 hours. The digested PCRproduct was ligated into pBluescript KS II(+) as described above and wasused for constructing the expression vector. The recombinant expressionvector containing DNA which codes TPO-[F141V] was named Refficacin-4 andwas deposited at the KCCM (Korean Culture Center of Microorganisms)under the Budapest Treaty on Jun. 9, 2003. Accession number given byinternational depositary authority was KCCM-10501.

C. Construction of cDNAs Coding G-CSF Muteins

Muteins of G-CSF, G-CSF[F13V], G-CSF[F83V], G-CSF[F113V], G-CSF[F140V],G-CSF[F144V] and G-CSF[F160V] were constructed according to proceduresas follows to have a single amino acid-substitution from phenylalanineto valine at each positions, respectively.

TABLE 3 Primers used in constructing cDNAs coding G-CSF- wild type and muteinsPrimer No. Nucleotide sequence Sequence No, 21 wild G-CSF Sense5.-CCCCGGGACCATGGCTGGACCTGCCACCCAG-3′ 38 22 Antisense5′-CGAATTCGCTCAGGGCTGGGAAGGAG-3′ 39 23 G-CSF-[F13V] Sense T7 24Antisense S-ACTTGAGCAGGACGCTCT-31 40 25 G-CSF-[F83V] Sense5′-AGCGGCCTTGTCCTCTA-3′  41. 26 Antisense T3 27 G-CSF-[F113V] Sense5′-GACGTTGCCACCACCAT-3′ 42 28 Antisense T3 29 G-CSF[F140V] Sense5′-GCCGTCGCCTCTGCTTT-3′ 43 30   Antisense 73 31 G-CSF[F144V] Sense5′-TCGCCTTCTGCTGTCCAG-3′ 44 32 Antisense T3 33 G-CSF-[F160V] Sense5′-TCTGCAAGACGTCCTGG-3′ 45 34 Antisense T3

Construction procedure of cDNA coding G-CSF muteins was basicallysimilar to that of TPOs. cDNAs which code G-CSF-[F13V], G-CSF-[F83V],G-CSF-[F113V], G-CSF-[F140V], G-CSF-[F144V], and G-CSF-[F160V] wereconstructed by primary PCR using specific primers (Table 3) anduniversal primer T3 and secondary PCR using the primary PCR product anduniversal primer T7. The template for these reactions was the cDNAcoding wild type G-CSF cloned in pBluescript KS II(+) obtained from theExample 1.

Mg²⁺ concentration was adjusted to 1 mM in the primary PCR. Sizes ofamplified megaprimers were 600 b.p for G-CSF-[F13V], 390 b.p forG-CSF-[F83V], 300 b.p for G-CSF-[F113V], 200 b.p for G-CSF-[140V], 200b.p for G-CSF-[F144V], and 150 b.p for G-CSF[F160V]. In the secondaryPCR using the megaprimers, cDNAs coding each muteins were amplified asthe same size of 640 b.p. Substitution from phenylalanine to valine atnucleotide sequence of the individual G-CSF mutein was verified bydirect sequencing.

Each PCR product of 640 b.p was separated in 0.8% agarose gel andpurified with Qiaex II gel extraction kit. The PCR product was digestedwith 15 U SmaI and IOU EcoRI at 37° C. for 2 hours and separated in 0.8%agarose gel and purified with Qiaex II gel extraction kit. The digestedPCR product was ligated into pBluescript KS II(+) as described above.The recombinant expression vector containing DNA which codesG-CSF-[F140V] was named Grefficacin4 and was deposited at the KCCM(Korean Culture Center of Microorganisms) under the Budapest Treaty onMay 17, 2004. Accession number given by international depositaryauthority was KCCM-10571.

D. Construction of cDNAs Coding GH Muteins

Four muteins of GEL GH-[F44V], GH-[F97V], GH-[F139V], GH-[F146V],GH-[F166V], and GH-[F176V] were constructed according to procedures asfollows to have a single amino acid-substitution from phenylalanine tovaline at each positions, respectively.

TABLE 4 Primers used in constructing cDNAs coding GH- wild type and muteinsPrimer No. Nucleotide Sequence Sequence No. 35 Leader Sense-15′-CTTTTGGCCTGCTCTGCCTGTCCTGGCTTCAA 46 sequenceGAGGGCAGTGCCTPCCCAACCATTCCCTTATC-3′ 36 addition Antisense T3 37 Sense-25′-GGAATTCATGGCTGCAGGCTCCCCGGACGTCC 47 CTGCTCCTGGCTTTTGGCCTGCTCTGCCT-3′38 Antisense T3 39 GH-[F44V] Sense T7 40 Antisense5′-GGGGTTCTGCAGGACTGAATACTTC-3′ 48 41 GH-[F97V] Sense T7 42 Antisense5′-GGCTGTTGGCGACGATCCTG-3′ 49 43 GH-[F139V] Sense T7 44 Antisense5′-GTAGGTCTGCTTGACGATCTGCCCAG-3′ 50 45 GH-[F146V] Sense T7 46 Antisense5′-GAGTTTGTGTCGACCTTGCTGTAG-3′ 51 47 GH-[F166V] Sense T7 48 Antisense5′-GTCCTDCCTGACGCAGTAGAGCAG-3′ 52 49 GH-[F176V] Sense T7 50 Antisense5′-CGATGCGCAGGACTGTCTCGACCTTGTC-3′ 53

Construction procedure of cDNA coding GH muteins was basically similarto that of TPOs. cDNAs which code muteins GH-[F44V], GH-[F97V],GH-[F139V], GH-[F146V], GH-[F166V] and GH-[F176V] were constructed byprimary PCR using specific primers (Table 4) and universal primer T3 andsecondary PCR using the primary PCR product and universal primer T7. Thetemplate for these reactions was the cDNA coding wild type GH cloned inpBluescript KS II(+) obtained from Example 1.

Mg²⁺ concentration was adjusted to 1 mM in the primary PCR. Sizes ofeach amplified megaprimers were 130 b.p for GH-[F44V], 300 b.p forGH-[F97V], 420 b.p for GH-[F139V], 450 b.p for GH-[F146V], 500 b.p forGH-[F166V] and 530 b.p for GH-[F176V] PCRs. Substitution fromphenylalanine to valine at nucleotide sequence of the individual GHmutein was verified by direct sequencing.

Each PCR product of 650 b.p was separated in 0.8% agarose gel andpurified with Qiaex II gel extraction kit. The PCR product was digestedwith 15 U EcoRI and 10 U HindIII at 37° C. for 2 hours and separated in0.8% agarose gel and purified with Qiaex II gel extraction kit. Thedigested PCR product was ligated into Bluescript KS II(+) as describedabove.

Example 3 Expression and Purification of TPO Muteins

A. TPO Muteins

a. Establishments of Transfected Cell Lines by Using Lipofection Method

Chinese hamster ovary (“CHO-K1”) (ATCC, CCL61) cells were prepared at adensity 1.5×10⁵ cells per 35 mm dish containing Dulbecco's modifiedEagle's medium (“DMEM”) [Gibco BRL, USA] supplemented with 10% fetalbovine serum (“FBS”). The cells were grown at 37° C. in a 5% CO₂ for18-24 hrs. 6 μl of Lipofectamine was added to 1.5 μg of the recombinantexpression vector comprising DNA coding TPO mutein in a sterile tube.Volume of this mixture was adjusted to 100 μl by adding serum-free DMEM.The tube was incubated at from temperature for 45 min. The cells grownin 35 mm dish were washed twice with serum-free DMEM and 800 μl ofserum-free DMEM was added to the dish. The washed cells were gentlyoverlaid on the lipofectamine-DNA complex and then incubated for 5 hrsat 37° C. in 5% CO₂. After 5 hrs incubation, 1 ml of DMEM containing 20%FBS was added to transfected cells and then the cells were incubated for18-24 hrs at 37° C., 5% CO₂. After the incubation, the cells were washedtwice with serum-free DMEM and then 2 ml of DMEM containing 10% FBS wasadded to the culture. These cells were incubated for 72 hrs at 37° C.,5% CO₂.

b. Analysis of Expression Level of TPO Muteins using ELISA

The cells transfected with plasmid containing cDNA coding TPO-wild typeor muteins were analyzed on their protein expression level by usingELISA assay. An goat anti-human TPO polyclonal antibody (R&D, USA)diluted to 10 μg/ml with coating buffer [0.1M Sodium bicarbonate, (pH9.6)] was added into each wells of 96 well plate (Falcon, USA) up to 100μl per well and incubated for 1 hour at room temperature. The plate waswashed with 0.1% Tween-20 in 1×PBS (PBST) three times. After washing,the plate was incubated with 200 μl of blocking buffer (1% FBS, 5%sucrose, 0.05% sodium azide) for 1 hour at room temperature and thenwashed three times with PBST. The cultured supernatants (including thetransfected cells) and dilution buffer [0.1% BSA, 0.05% Tween-20, 1×PBS]were mixed with serial dilutions. 25 ng/ml of recombinant human TPO[Calbiochem, USA] as a positive control and untransfected CHO-K1cultured supernatants as a negative control were equally diluted. Thesecontrols and samples were incubated for 1 hr at room temperature. Then,the plate was washed with PBST three times. A biotinylated goatanti-human TPO antibody (R&D, USA) diluted to 0.2 μg/ml with dilutionbuffer was added to the 96 well plate up to 100 μl per well andincubated for 1 hr at room temperature. The plate was washed with PBSTthree times. Streptavidin-HRP (R&D, USA) diluted to 1:200 in dilutionbuffer was added 100 μl per well to the 96 well plate and incubated for1 hr at room temperature. After 1 hour, the plates was washed threetimes with PBST, and then coloring reaction was performed by using TMBmicrowell peroxidase substrate system (KPL, USA) and OD) was read at 630nm with microplate reader [BIO-RAD, Model 550].

c. Analysis of Expression Level and Molecular Weight of Mutein TPO UsingWestern Blotting

In order to exclude FBS in medium, CHO-S-SFM II (Gibco BRL, USA) wasused for culture of the above-transfected cell. Culture medium fromCHO-S-SFM II was filtrated with 0.2 μm syringe filter and concentratedwith centricon (Mol. 30,000 Millipore, USA). To perform the reducedSDS-PAGE, sample-loading buffer containing 5% β-mercaptoethanol wasadded to the sample and heated for 5 minutes. Stacking gel and runninggel were used for this SDS-PAGE. The stacking gel was composed of 3.5%acrylamide, 0.375 M Tris (pH6.8), 0.4% SDS and the miming gel wascomposed of 10% acrylamide gel, 1.5 M Tris (pH0.8), 0.4% SDS. AfterSDS-PAGE gel running treatment, protein samples were transferred toWestern (PVDF transfermembrane, S&S) having 4 μm pore at 350 mA for 2hrs in a 25 mM Tris-192 mM glycine (pH 8.3)-20% methanolbuffer-containing reservoir. After transferring, it was blocked threetimes for 10 minutes with 5% fat free milk powder in PBST. Thebiotinylated goat anti-human TPO antibody (R&D, USA) was diluted to 0.25μg/ml in blocking buffer and 3 ml of this solution was added and shakenfor 6 hrs. The membrane was washed with washing solution three times.Streptavidin-HRP (R&D, USA) was diluted to 1:100 in blocking buffer andincubated for 1 hr. The membrane was washed three times with washingsolution. Protein bands were visualized by incubating with DAB substrate(VECTOR LABORATORIES, USA) for 10 minutes. This reaction was stoppedwith soaking the membrane in deionized water.

In FIG. 2 a, wild type and mutein forms of TPOs had the same molecularweight (55 kD).

Relative expression level of wild type and muteins of TPO was shown inFIG. 3 a. Expression level of each TPO mutein was compared to that ofwild type TPO as a control. Expression level of TPO-[F128V] wasincreased 1.4 times more than that of wild type TPO. But expressions ofTPO-[F46V], -[F131V] and -[F141V] were decreased to 20%, 40%, and 40% ofthat of wild type, respectively.

B. EPO Muteins

Expression vectors containing cDNAs coding EPO muteins were transfectedto CHO-K1 cell and expression level of each of EPO mutein was detectedby using ELISA assay. And molecular weight of each of wild type andmutein of TPO was analyzed by western blotting.

In FIG. 2 b, wild type and mutein forms of EPO had the same molecularweight (45 kD).

Relative expression level of wild type and muteins of EPO was shown inFIG. 3 b. Expressions level of EPO-[F48V] and -[F138V] was increased 1.4and 1.2 times more than that of the wild type EPO, respectively. Butexpression level of EPO-[F142V] and -[F148V] was decreased to 20% and30% of that of wild type EPO, respectively.

C. G-CSF Muteins

Expression vectors containing cDNAs coding G-CSF muteins weretransfected to CHO-K1 cell and expression level of each G-CSF mutein wasdetected by using ELISA assay. And molecular weight of each of wild typeand muteins of G-CSF was analyzed by western blotting.

In FIG. 2 c, wild type and mutein forms of G-CSF had the same molecularweight (50 kD).

Relative expression level of wild type- and muteins of G-CSF was shownin FIG. 3 c. Expression levels of rest of G-CSF muteins were similar tothat of wild type G-CSF. Expression level of G-CSF mutein-[F83V] wasincreased 1.9 times than that of wild-type. But expression levels ofG-CSF muteins-[F140V] and -[F144V] were decreased to 50% and 70% of thatof wild type G-CSF, respectively.

D. GH Muteins

Expression vectors containing cDNAs coding GH muteins were transfectedto CHO-K1 cell. Method for the expression of each of the GH muteins wasthe same as those used for TPO production.

Example 4 Construction of DNA Coding EPO, TPO, G-CSF, and GH Receptors

A. Construction of DNA Coding EPO and TPO Receptors

DNAs coding EPO and TPO receptors were constructed to analyze bindingaffinities of each of EPO muteins and TPO muteins. DNA codingextracellular domain of each receptor was linked to DNA coding Fc domainof IgG1 such that the C-terminal region of extracellular domain of eachreceptor was fused to N-terminal region of human IgG1 Fc domain. cDNAcoding EPO receptor was constructed by PCR using sense primer (primer51) with restriction sites of EcoRI and leader sequence of EPO receptorand antisense primer (primer 52) with the sequence coding 3′ end of EPOreceptor and the sequence coding 5′ end Fc domain of IgG. cDNA codingTPO receptor linked to Fe domain of IgG1 was constructed by PCR usingsense primer (primer 53) with restriction sites of HindIII and leadersequence of TPO receptor and antisense primer (primer 54) with thesequence coding 3′ end of TPO receptor and the sequence coding 5′ end ofFc domain of IgG.

cDNA coding EPO receptor produced as described above and DNA coding Fcdomain of IgG1 were mixed in the same tube, complementary bindingbetween the common sequences was induced. Using this mixture, cDNAcoding EPO receptor linked to Fc domain of IgG1 was constructed by PCRusing sense primer (primer 51) with restriction sites of EcoRI andleader sequence of EPO receptor and antisense primer (primer 55) withrestriction sites of XbaI and 3′ end of Fc domain of IgG. The PCRproduct was cut with HindIII and XbaI and inserted into PCR-3 expressionvector for production of EPO receptor-Fc fusion protein.

TABLE 5  A List of primers used in constructing EPO and EPO receptorsfused to immunoglobulin Primer Sequence No. Nucleotide sequence No. EPO51 Sense 5′-CGGAATTCATGGACCACCTCGGGGCG-3′ 54 receptor 52 Antisense5′-GCTCTAGACTAAGAGCAAGCCACATAGCTGGG-3′ 55 TPO 53 Sense5′-CCCAAGCTTATGGAGCTGACTGAATTGCTCCTC-3′ 56 receptor 54 Antisense5′-GGAATTCTTACCCTTCCTGAGACAGATTCTGG-3′ 57 IgGI-R- 555′-GCTCTAGAGCTCATTTACCCGGAGACAGGGAGAG-3′ 58 XbaI

B. Construction of DNA Coding G-CSF and GH Receptors

cDNA coding G-CSF receptor was constructed by PCR using sense primer(primer 56) with restriction site of HindIII and leader sequence ofG-CSF receptor and antisense primer (primer 57) with restriction site ofEcoRI and the sequence coding 3′ end of G-CSF receptor. cDNA coding GHreceptor was constructed by PCR using sense primer (primer 58) withrestriction site of EcoRI and leader sequence of G-CSF receptor andantisense primer (primer 59) with restriction site of SpeI and thesequence coding 3′ end of G-CSF receptor.

The PCR product encoding G-CSF receptor was digested with HindIII andEcoRI, and was cloned by inserting into a commercially available cloningvector, pBluescript KS II(+) at HindIII/EcoRI site. The PCR productencoding GH receptor was digested with EcoRI and SpeI, and cloned byinserting into a commercially available cloning vector, pBluescript KSII(+) at EcoRI/SpeI site.

Fc domain of human IgG was constructed by PCR using sense primer(printer 60 for G-CSF, primer 61 for GH) with sequence coding 5′ endpart of hinge region of human IgG and antisense primer (primer 62). ForG-CSF receptor, the PCR product coding Fc domain of human IgG wasdigested with EcoRI and XbaI, and cloned by inserting into acommercially available cloning vector, pBluescript KS II(+) atEcoRI/XbaI site. For GH receptor, the PCR product coding Fc domain ofhuman IgG was digested with SpeI and XbaI, and cloned by inserting intoa commercially available cloning vector, pBluescript KS II(+) at SpeIsite/XbaI.

Both of the cloned cDNA coding G-CSF receptor and the cloned Fc domainof human IgG were digested with EcoRI/XbaI and then ligated to prepareDNA coding G-CSF receptor linked to Fc domain of human IgG. This DNAconstruct was cut with HindIII and XbaI and inserted into PCR-3expression vector. Both of the cloned cDNA coding GH receptor and thecloned Fc domain of human IgG were digested with SpeI/XbaI and thenligated to prepare DNA coding G-CSF receptor linked to Fc domain ofhuman IgG. This DNA construct was cut with EcoRI and XbaI and insertedinto PCR-3 expression vector.

TABLE 6  A List of primers used in constructing G-CSF andGH receptors fused to Immunoglobulin Primer Sequence No.Nucleotide sequence No. G-CSF 56 Sense5′-CCCAAGCTTATGGCTGGACCTGCCACCC-3′ 59 receptor 57 Antisense5′-GGAATTCGCAACAGAGCCAGGCAGTMCA-3′ 60 GH 58 Sense5′-CGGAATTCATGGATCTCTGGCAGCTG-3′ 61 receptor 59 Antisense5′-GGACTAGTTTGGCTCATCTGAGGAAGTG-3′ 62 IgG1-F 60 Sense5′-GGAATTCGCAGAGCCCAAATCTTGTGACAAAACTC-3′ 63 EcoR 1 IgG1-F- 61 Sense5′-GACTAGTGCAGAGCCCAAATCTTGTGA-3′ 64 Spe I IgGI-R- 62 Antisense5′-GCTCTAGAGCTCATTTACCCGGAGACAGGGAGAG-3′ 65 Xbal

Example 5 Measurement of Binding Affinity of Cytokines and their Muteinsto Each of their Receptors by Using ELISA

A. Binding of TPO and TPO Muteins to TPO Receptor

Culture supernatants of CHO cell transfected with expression vectorscarrying genes for TPO muteins were used for measuring cytokine-receptorinteractions.

TPO receptor-Ig fusion protein was purified from culture supernatant ofCHO cell transfected with recombinant expression vector carrying genecoding for TPO receptor-Fc fusion protein by using Protein ASepharose-4B column (Pharmacia, Sweden). The purified fusion proteindiluted to 10 μg/ml with coating buffer [0.1M Sodium bicarbonate, (pH9.6)] was added into each wells of 96 well plate (Falcon, USA) up to 100μl per well and incubated for 1 hour at room temperature. The plate waswashed with 0.1% Tween-20 in 1×PBS [PBST] three times. After washing,the plate was incubated with 200 μl of blocking buffer (1% FBS, 5%sucrose, 0.05% sodium azide) for 1 hour at room temperature and thenwashed three times with PBST.

After washing, culture supernatants consisting of four TPO muteins andone TPO wild type, respectively were diluted serially with dilutionbuffer [0.1% BSA, 0.05% Tween-20, 1×PBS] and was added to 96 well platecoated with the TPO receptor-Fc fusion protein and incubated for 1 hr.The washing was repeated three times with PBST. A recombinant human TPO[Calbiochem, USA] as a positive control, and untransfected CHO-K1cultured supernatants as a negative control were equally diluted. Theplates were washed with PBST three times. A biotinylated goat anti-humanTPO antibody (R&D, USA) diluted to 0.2 μg/ml in dilution buffer wasadded to the 96 well plate to 100 μl per well and incubated for 1 hr atroom temperature. The plate was washed with PBST three times.Streptavidin-HRP (R&D, USA) diluted to 1:200 in dilution buffer wasadded 100 μl per well to 96 well plate and incubated for 1 hr at roomtemperature. The plate was washed three times with PBST after 1 hour.Coloring reaction was performed using TMB microwell peroxidase substratesystem (KPL, USA) and O.D was read at 630 nm with microplate reader[BIO-RAD, Model 550].

The binding affinity of TPO-[F141V] and TPO-[F131V] to the TPO receptorwas increased compared to that of wild type TPO (FIG. 4 a). And theformer mutein had the strongest binding affinity among all TPO muteins.

B. Binding of EPO and EPO Muteins to EPO Receptor

Measurement of binding affinity of EPO wild type and muteins to thereceptor was basically similar to that of binding affinity of TPO andTPO muteins to TPO Receptor.

The binding affinity of EPO-[F148V] and EPO-[F142V] to the EPO receptorwas increased compared to that of wild type EPO (FIG. 4 b). And theformer mutein had the strongest binding affinity among all EPO muteins.

C. Binding of G-CSF and G-CSF Muteins to G-CSF Receptor

Measurement of binding affinity of G-CSF wild type and muteins to thereceptor was basically similar to that of binding affinity of TPO andTPO muteins to TPO Receptor.

Results (FIG. 4 c) showed binding affinity of G-CSF-[F140V],G-CSF-[F144V], and G-CSF-[F160V] to the G-CSF receptor was increasedcompared to that of wild type G-CSF. And the first mutein(G-CSF-[F140VD]) had the strongest binding affinity among all G-CSFmuteins.

D. Binding of GH and GH Muteins to GH Receptor

Measurement of binding affinity of GH wild type and muteins to thereceptor was basically similar to that of binding affinity of TPO andTPO muteins to TPO Receptor.

Results (FIG. 4 d) showed that GH-[F139V] had the strongest bindingaffinity to the GH receptor.

Example 6 Measurement of Bindings of Cytokines and their Muteins to Eachof their Receptors by Using SPR

A. Binding of TPO and TPO Muteins to TPO Receptor

To measure the binding affinity of TPO-[F141V] and TPO-[F131V] to TPOreceptor, SPR was performed on a BIAcore 3000 instrument containing CM5sensor chip. Anti-human IgG antibody was immobilized onto each flowcells 1 and 2 using amine-coupling chemistry. To inactivate any activegroup, surfaces were blocked with 1 M ethanolamine. TPO receptor-Fcfusion protein was added to bind to the anti-human IgG antibody for 2min at 30 μl/min and then TPO and TPO muteins were reacted to bind tothe TPO receptor.

At the same density of ligand, increased resonance unit (RU) meanshigher binding affinities. In FIG. 5 a, wild TPO, TPO-[F141V] andTPO-[F131V] were 10RU, 30RU and 20RU, respectively. This result showedthat TPO-[F141V] had the strongest binding affinity. In addition, K_(D)values of wild type and mutein TPO were shown in Table 7.

TABLE 7 Changes of Binding-kinetic rate constant of wild type and muteinTPO Relative K_(on)(M⁻¹s⁻¹) × K_(off)(S⁻¹) × K_(D)(μM) = Binding 10⁵10⁻² K_(off)/K_(on) Chi² affinity Wild 2.42 13.7 5.66 5.81 1 type TPOTPO- 12.8 0.51 0.04 6.03 141 [F141]

B. EPO Muteins

SPR was performed to measure binding affinities of EPO mutein-[F148V]and EPO-[F142V] with EPO receptor. Experimental procedure was similar tothat for TPOs.

FIG. 5 b was the SPR result of EPO wild type and muteins. In FIG. 5 b,EPO-[F148V] showed 40RU, EPO-[F142V] 30RU. These results show thatEPO-[F148V] had the strongest binding affinity. In addition, K_(D)values of EPO muteins were shown in Table 8.

TABLE 8 Changes of Binding-kinetic rate constant of wild type and muteinEPO Relative K_(on)(M⁻¹s⁻¹) × K_(off)(S⁻¹) × K_(D)(μM) = Binding 10⁵10⁻² K_(off)/K_(on) Chi² affinity Wild 1.84 8.83 4.80 4.55 1 type EPOEPO- 14.0 0.64 0.05 2.26 105 [F148]

Example 7 Measurement of Binding Affinities of Wild Type- and Muteins ofCytokine by Using FACS

A. Establishment of TF-1/c-Mpl Cell Line

TF-1/c-Mpl cell line was established by transfecting cDNA coding c-Mplinto T-1 cell. Expression of c-Mpl was verified by using FACS analysis.The 1×10⁶/ml of the TF-1/c-Mpl cells was washed with PBS buffer andpurified c-Mpl mouse anti-human monoclonal antibody (BD PharMingen, USA)was incubated with the TF-1/c-Mpl cells. And then FTIC-conjugatedanti-mouse IgG (whole molecule; Sigma, USA) was added to verifyexpression of c-Mpl on surface of the TF-1/c-Mpl cells. As a result,graph of the TF-1/c-Mpl cell was shifted rightward from that of TF-1cells. This result showed that c-Mpl, TPO receptor, was expressed on theTF-1/c-Mpl cell.

B. FACS Analysis of TPO Muteins

The 1×10⁶/ml of TF-1/c-Mpl cell was suspended in PBS buffer and TPO wildtype and -[F141V] was added to the suspension and incubated at 4° C. for30-60 minutes, respectively. Biotinylated goat anti-human TPO polyclonalantibody (R&D, USA) was added to the cells above and incubated at 4° C.for 30-60 minutes. Streptavidin-FITC (Sigma, USA) was added to the cellsabove and incubated at 4° C. for 30-60 minutes. The cells were washedtwice with PBS buffer to remove non-reacted Streptavidin-FITC. The cellswere suspended in PBS buffer and flow cytometric analysis was performedat 488 nm using EXCALIBUR (BD, U.S.A.).

In FIG. 6 a, a binding curve of TPO-[F141V] was shifted rightward fromthat of wild type TPO. This result showed that TPO-[F141V] had muchstronger receptor-binding affinity than the wild type TPO.

C. FACS Analysis of EPO Muteins

FACS procedure of EPO muteins was carried out similarly to that of TPO.

In FIG. 6 b, a binding curve of EPO-[F148V] was shifted rightward fromthat of wild type TPO. This result showed that TPO-[F141V] is muchstronger in receptor-binding affinity than the wild type EPO.

Example 8 Measurement of Biological Activities of TPO, EPO, G-CSF and GHMuteins

A. Cell Proliferation Assay of TPO Muteins

To investigate differences of cell proliferation and biologicalactivities between TPO-wild type and muteins, TF-1/c-Mpl cell lineproduced above was used. TF-1/c-Mpl cells were grown in DMEM mediumsupplemented with 10% fetal bovine serum, 1 ng/ml GM-CSF at 37° C., 5%CO₂. 0.4, 1, 5, 10, 20, 40, 75 ng/ml, of each of TPO-wild type andmuteins in RPMI-1640 were seeded in 96-well tissue-culture plates(FALCON, USA). 1×10⁴ cell of the TF-1/c-Mpl cells in RPMI-1640containing 10% fetal bovine serum was added to each wells of the 96-wellplate. After 4 days cultivation at 37° C., 5% CO₂, 20 μl of MTS solution[3-(4,5-dimethyl-2-yl)-5-(3-arboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium,inner salt, MTS] and the phenazine ethosulfate (PES; promega) was addedand incubated for 4 hours. O.D. was measured with microplate reader(BIO-RAD Model 550) at 490 nm.

FIG. 7 a showed differences of TPO wild type and muteins in stimulatingTF-1/c-Mpl cell proliferation. TPO was applied to the TF-1/c-Mpl from0.4 ng/ml to 75 ng/ml. Cell proliferation was increased up to 50 ng/mlof TPO concentration. TF-1/c-Mpl cell proliferation potential ofTPO-[F141V] was much stronger than that of wild type and was the firstin biological activity among TPO muteins. Biological activity ofTPO-[F131V] was the second strongest among TPO muteins. Activity ofTPO-[F46V] was similar to that of wild type.

B. Cell Proliferation Assay of EPO Muteins

Biological activity for EPO muteins was examined by cell proliferationassay using EPO-dependent TF-1 cell. Experimental procedure of cellproliferation assay of EPO muteins was similar to that of TPO muteins.

FIG. 7 b showed differences of EPO wild type and muteins in stimulatingTF-1 cell proliferation FPO was applied to the TF-1 Cell from 0.01 IU/mlto 7 IU/ml. TF-1 cell proliferation potential of EPO-[F148V] was muchstronger than that of the wild type and was the first in biologicalstrength among EPO muteins. Biological activities of EPO-[F142V] andEPO-[F138V] were the second and the third strongest among EPO muteins,respectively.

TABLE 9 Biological activities of TPOs The maximum activity TPOcomparison(%) Wild type 100 Muteins TPO-[F46V] 107 TPO-[F128V] 63TPO-[F131V] 119 TPO-[F141V] 146

TABLE 10 Biological activities of EPOs The maximum activity EPOcomparison(%) Wild type 100 Muteins EPO-[F48V] 84 EPO-[F138V] 57EPO-[F142V] 122 EPO-[F148V] 137

C. G-CSF Muteins

Biological activity for G-CSF muteins was examined by cell proliferationassay using G-CSF dependent HL-60 cell. Experimental procedure of cellproliferation assay of G-CSF muteins was similar to that of TPO muteins.

FIG. 7 c showed differences of G-CSF wild type and muteins instimulating HL-60 cell proliferation. G-CSF was applied to the HL-60Cell from 0.4 ng/ml to 75 ng/ml. HL-60 cell proliferation potential ofG-CSF-[F140V] was much stronger than that of the wild type and was thefirst in biological strength among G-CSF muteins.

D. GH Muteins

Biological activity for GH muteins was examined by cell proliferationassay using GH dependent NB2 cell. Experimental procedure of cellproliferation assay of GH muteins was similar to that of GH muteins.

FIG. 7 d showed differences of GH wild type and muteins in stimulatingNB2 cell proliferation. GH was applied to the NB2 Cell from 0.4 ng/ml to75 ng/ml. NB2 cell proliferation potential of GH-[F139V] was muchstronger than that of the wild type and was the first in biologicalstrength among GH muteins.

Example 9 Pharmacokinetic Profiles of EPO- and TPO-wild types andmuteins

Difference of pharmacokinetic profiles of each EPO- and TPO-muteinsbetween their wildtype was investigated. TPO or TPO muteins was injectedintravenously into rabbits (New Zealand White, 3 kg). And then bloodsamples were collected serially. EPO and TPO concentrations from eachsamples were detected by using quantitative ELISA assay as describedabove. Injection of EPOs into mice (12 weeks, Balb/c, 30 g) wasperformed by both intraperitonealy and intravenously. Blood samples inheparin-containing tubes were separated by centrifugation at 3,000 rpmfor 10 minutes. Supernatant containing plasma was used to detect bloodconcentrations of EPO and TPO by using ELISA.

After intravenous injection of 5 μg/kg of TPO wild type and -[F141V]into rabbit, plasma concentration profiles of TPO wild type and -[F141V]were shown in FIG. 8 a. Concentration of TPO-[F141V] was decreased morerapidly than that of wild type TPO. TPO-[F141V] was shifted from bloodto peripheral target tissues more rapidly, due to its stronger bindingaffinity to receptor.

After intravenous injection of 1000 I.U/kg of wild type EPO andEPO-[F148V] into rabbit, plasma concentration profiles of wild type EPOand EPO-[F148V] in blood were shown in FIG. 8 b. Concentration ofEPO-[F148V] was decreased more rapidly than that of EPO wild type.

After intraperitoneal injection of 20 I.U/g of wild type EPO andEPO-[F148V] into mice, plasma concentration profiles were shown in FIG.8 c. The diffusion velocity of EPO wild type was higher than that ofEPO-[F148V] at early stage and maximum concentration in blood (Cmax) ofwild type EPO was also higher than that of EPO-[F148V]. Cmax ofEPO-[F148V] remained longer than wild type EPO. These results suggestedthat EPO-[F148V] was more hydrophobic and had higher binding affinity toreceptor than the wild type EPO. And these results lead to theconclusion that EPO-[F148V] was diffused into blood more slowly andshifted from blood to peripheral target tissues more quickly than thoseof wild type FPO.

TABLE 11 Pharmacokinetic parameters of EPO wild type and EPO-[F148V]mutein Mouse Rabbit Wild type EPO-mutein Wild type EPO-mutein EPO[F148V] EPO [F148V] T_(1/2)(Half life) 1.9 1.4 3.8 2.4 AUC 100 78 100 80

Example 10 In Vivo Activities of EPO Muteins

Difference of biological activities between EPO-wild type and muteinswas verified in mice. Mice (12 weeks Balb/c, 20 g, Jungang Lab AnimalInc., Korea) were γ-irradiated at 700Rad. 250 ng of purified EPO wildtype and muteins in 50 μl of PBS were injected intraperitoneally 3 timeseveryday. Blood samples were collected from their tail vein. And thenhematologic parameters were tested according to ordinary CBC test. Wildtype EPO was used as a positive control and CHO cell culture supernatantwas used as a negative control. Blood was collected into tubescontaining EDTA at 0, 1st, 2nd, 4th, 7th, 10th, 15th, 20th, 25th, and30th days after the injection.

FIG. 9 showed that CBC results in mice injected intraperitoneally withEPO-wild type and muteins to verify change in count of RBC andreticulocyte. Increase of RBC count (FIG. 9 a) was much more remarkablein EPO-[F148V]-injected mice than mice injected with wild type EPO. Andthe RBC increase of in EPO-[F48V]- and EPO-[138V]-injected mice wasweaker than that of mice injected with wild type EPO. Increase ofreticulocyte count (FIG. 9 b) and hematocrit was similar to the resultof RBC count change in mice injected with EPO-[F148V].

Example 11 In Vivo Activities of TPO Muteins

Difference of biological activities between TPO-wild type and muteinswas studied in mice. Mice (12 weeks Balb/c, 20 g, Jungang Lab AnimalInc., Korea) were γ-irradiated at 700Rad. 250 ng of purified TPO wildtype and muteins in 50 μl of PBS were injected intraperitoneally 3 timeseveryday. Blood samples were collected from their tail vein. And thenhematologic parameters were tested according to ordinary CBC test. Wildtype TPO was used as a positive control and CHO cell culture supernatantwas used as a negative control. Blood was collected into tubescontaining EDTA at 0, 1st, 4th, 7th, 10th, 14th, 18th 23rd, 28th, and32nd days after injection.

FIG. 10 showed the changes of platelet count (FIG. 10 a), leukocytecount (FIG. 10 b), and neutrophil count (FIG. 10 c) in mice injectedintraperitoneally with TPO-wild type and muteins. Increase of plateletcount was the most remarkable in mice injected with TPO-[F141\7]. Andmice injected with TPO-[F131V] was the second highest. Mice injectedwith TPO-[F46V] was similar to those injected with wild type TPO. Andmice injected with TPO-[F128V] showed platelet count similar to that ofnegative controls injected with PBS (FIG. 10 a). Increase of leukocytecount (FIG. 10 b) and neutrophil count (FIG. 10 c) showed similarpatterns as those seen in platelet change.

INDUSTRIAL APPLICABILITY

As apparent from the above results of the present invention, valinesubstitution for phenylalanine residue, which is present in a domainparticipating in the binding of conventional wild-type biologicalresponse-modulating proteins to corresponding receptors, ligands orsubstrates, leads to an increase in binding affinity and biologicalactivity, and reduces the production of autoantibodies to conventionalprotein variants, thereby making it possible to produce improved proteindrugs.

Indications Relating to Deposited Microorganism or Other BiologicalMaterial PCT Rule 13bis

A. The indications made below relate to the deposited microorganism orother biological material referred to in the description on page 38,line 20-26 B. IDENTIFICATION OF DEPOSIT Further deposits are on anadditional sheet□ Name of depositary institution Korean Culture Centerof Microorganisms(KCCM) Address of depositary institution(includingpostal code and country) 361-221, Yurim B/D, Hongje-dong, Seodaemun-gu,SEOUL 120-091, Republic of Korea Date of deposit Accession Number 09Jun. 2003 KCCM - 10500 C. ADDITIONAL INDICATIONS(leave blank if notapplicable) This information is continued on an additional sheet □ D.DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE(if the indications arenot for all designated States) E. SEPARATE FURNISHING OFINDICATIONS(leave blank if not applicable) The indications listed belowwill be submitted to the International Bureau later(specify the generalnature of the indications e.q., “Accession Number of Deposit”) Forreceiving Office use only For international Bureau use only □ This sheetwas received with the international □ This sheet was received by theInternational application Bureau on: Authorized officer Authorizedofficer Form PCT/RO/134(July 1998)

Indications Relating to Deposited Microorganism or Other BiologicalMaterial PCT Rule 13bis

A. The indications made below relate to the deposited microorganism orother biological material referred to in the description on page 40,line 1-10 B. IDENTIFICATION OF DEPOSIT Further deposits are on anadditional sheet□ Name of depositary institution Korean Culture Centerof Microorganisms(KCCM) Address of depositary institution(includingpostal code and country) 361-221, Yurim B/D, Hongje-dong, Seodaemun-gu,SEOUL 120-091, Republic of Korea Date of deposit Accession Number 09Jun. 2003 KCCM - 10501 C. ADDITIONAL INDICATIONS(leave blank if notapplicable) This information is continued on an additional sheet □ D.DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE(if the indications arenot for all designated States) E. SEPARATE FURNISHING OFINDICATIONS(leave blank if not applicable) The indications listed belowwill be submitted to the International Bureau later(specify the generalnature of the indications e.q., “Accession Number of Deposit”) Forreceiving Office use only For international Bureau use only □ This sheetwas received with the international □ This sheet was received by theInternational application Bureau on: Authorized officer Authorizedofficer Form PCT/RO/134(July 1998)

Indications Relating to Deposited Microorganism or Other BiologicalMaterial PCT Rule 13bis

A. The indications made below relate to the deposited microorganism orother biological material referred to in the description on page 41,line 10-15 B. IDENTIFICATION OF DEPOSIT Further deposits are on anadditional sheet□ Name of depositary institution Korean Culture Centerof Microorganisms(KCCM) Address of depositary institution(includingpostal code and country) 361-221, Yurim B/D, Hongje-dong, Seodaemun-gu,SEOUL 120-091, Republic of Korea Date of deposit Accession Number 17 May2004 KCCM - 10571 C. ADDITIONAL INDICATIONS(leave blank if notapplicable) This information is continued on an additional sheet □ D.DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE(if the indications arenot for all designated States) E. SEPARATE FURNISHING OFINDICATIONS(leave blank if not applicable) The indications listed belowwill be submitted to the International Bureau later(specify the generalnature of the indications e.q., “Accession Number of Deposit”) Forreceiving Office use only For international Bureau use only □ This sheetwas received with the international □ This sheet was received by theInternational application Bureau on: Authorized officer Authorizedofficer Form PCT/RO/134(July 1998)

1-9. (canceled)
 10. A protein variant, wherein a valine residue replacesa phenylalanine residue in a binding domain of a protein having abiological response-modifying function when bound to a receptor, ligand,or substrate.
 11. The protein variant according to claim 10, wherein theprotein is a cytokine.
 12. The protein variant according to claim 11,wherein the cytokine is a 4-alpha helix bundle cytokine.
 13. The proteinvariant according to claim 12, wherein the 4-alpha helix bundle cytokineis selected from the group consisting of CNTF, EPO, Flt3L, G-CSF,GM-CSF, GH, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12p35, LPT, LIF, M-CSF,OSM, PL, SCF, TPO, IFN-α2A, IFN-α2B, IFN-β, IFN-γ, IFN-ω, and IFN-τ. 14.The protein variant according to claim 13, wherein the 4-alpha helixbundle cytokine is CNTF, EPO, Flt3L, G-CSF, GM-CSF, GH, IL-2, IL-3,IL-4, IL-5, IL-6, IL-12p35, LPT, LIF, M-CSF, OSM, PL, SCF or TPO and thevaline residue replaces a phenylalanine residue at an amino acid residuebetween positions 110 and
 180. 15. The protein variant according toclaim 13, wherein the 4-alpha helix bundle cytokine is IFN-α2A, IFN-α2B,IFN-β, IFN-γ, IFN-ω or IFN-τ and the valine residue replaces thephenylalanine residue at an amino acid residue between positions 1 and50.
 16. The protein variant according to claim 13, wherein the 4-alphahelix bundle cytokine is CNTF and the valine residue replaces thephenylalanine residue at position 3, 83, 98, 105, 119, 152, or 178 ofSEQ ID NO.:
 1. 17. The protein variant according to claim 13, whereinthe 4-alpha helix bundle cytokine is EPO and the valine residue replacesthe phenylalanine residue at position 48, 138, 142, or 148 of SEQ IDNO.:
 2. 18. The protein variant according to claim 13, wherein the4-alpha helix bundle cytokine is Flt3L and the valine residue replacesthe phenylalanine residue at position 6, 15, 81, 87, 96, or 124 of SEQID NO.:
 3. 19. The protein variant according to claim 13, wherein the4-alpha helix bundle cytokine is G-CSF and the valine residue replacesthe phenylalanine residue at position 13, 83, 113, 140, 144, or 160 ofSEQ ID NO.:
 4. 20. The protein variant according to claim 13, whereinthe 4-alpha helix bundle cytokine is GM-CSF and the valine residuereplaces the phenylalanine residue at position 47, 103, 106, 113, or 119of SEQ ID NO.:
 5. 21. The protein variant according to claim 13, whereinthe 4-alpha helix bundle cytokine is GH and the valine residue replacesthe phenylalanine residue at position 1, 10, 25, 31, 44, 54, 92, 97,139, 146, 166, 176, or 191 of SEQ ID NO.:
 6. 22. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine is IL-2and the valine residue replaces the phenylalanine residue at position42, 44, 78, 103, 117, or 124 of SEQ ID NO.:
 13. 23. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine is IL-3and the valine residue replaces the phenylalanine residue at position37, 61, 107, 113, or 133 of SEQ ID NO.:
 14. 24. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine is IL-4and the valine residue replaces the phenylalanine residue at position33, 45, 55, 73, 82, or 112 of SEQ ID NO.:
 15. 25. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine is IL-5and the valine residue replaces the phenylalanine residue at position49, 69, 96, or 103 of SEQ ID NO.:
 16. 26. The protein variant accordingto claim 13, wherein the 4-alpha helix bundle cytokine is IL-6 and thevaline residue replaces the phenylalanine residue at position 73, 77,93, 104, 124, 169, or 172 of SEQ ID NO.:
 17. 27. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine isIL-12p35 and the valine residue replaces the phenylalanine residue atposition 13, 39, 82, 96, 116, 132, 150, 166, or 180 of SEQ ID NO.: 18.28. The protein variant according to claim 13, wherein the 4-alpha helixbundle cytokine is LPT and the valine residue replaces the phenylalanineresidue at position 41 or 92 of SEQ ID NO.:
 19. 29. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine is LIFand the valine residue replaces the phenylalanine residue at position41, 52, 67, 70, 156, or 180 of SEQ ID NO.:
 20. 30. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine isM-CSF and the valine residue replaces the phenylalanine residue atposition 35, 37, 54, 67, 91, 106, 121, 135, 143, 229, 255, 311, 439,466, or 485 of SEQ ID NO.:
 21. 31. The protein variant according toclaim 13, wherein the 4-alpha helix bundle cytokine is OSM and thevaline residue replaces the phenylalanine residue at position 56, 70,160, 169, 176, or 184 of SEQ ID NO.:
 22. 32. The protein variantaccording to claim 13, wherein the 4-alpha helix bundle cytokine is PLand the valine residue replaces the phenylalanine residue at position10, 31, 44, 52, 54, 92, 97, 146, 166, 176, or 191 of SEQ ID NO.:
 23. 33.The protein variant according to claim 13, wherein the 4-alpha helixbundle cytokine is SCF and the valine residue replaces the phenylalanineresidue at position 63, 102, 110, 115, 116, 119, 126, 129, 158, 199,205, 207, or 245 of SEQ ID NO.:
 24. 34. The protein variant according toclaim 13, wherein the 4-alpha helix bundle cytokine is TPO and thevaline residue replaces the phenylalanine residue at position 141, 186,204, 240, or 286 of SEQ ID NO.:
 25. 35. The protein variant according toclaim 13, wherein the 4-alpha helix bundle cytokine is IFN-α2A and thevaline residue replaces the phenylalanine residue at position 27, 36,38, 43, 47, 64, 67, 84, 123, or 151 of SEQ ID NO.:
 7. 36. The proteinvariant according to claim 13, wherein the 4-alpha helix bundle cytokineis IFN-α2B and the valine residue replaces the phenylalanine residue atposition 27, 36, 38, 43, 47, 64, 67, 84, 123, or 151 of SEQ ID NO.: 8.37. The protein variant according to claim 13, wherein the 4-alpha helixbundle cytokine is IFN-β and the valine residue replaces thephenylalanine residue at position 8, 38, 50, 67, 70, 111, or 154 of SEQID NO.:
 9. 38. The protein variant according to claim 13, wherein the4-alpha helix bundle cytokine is IFN-γ and the valine replaces thephenylalanine residue at position 18, 32, 55, 57, 60, 63, 84, 85, 95, or139 of SEQ ED NO.:
 10. 39. The protein variant according to claim 13,wherein the 4-alpha helix bundle cytokine is IFN-ω and the valinereplaces the phenylalanine residue at position 27, 36, 38, 65, 68, 124,or 153 of SEQ ID NO.:
 11. 40. The protein variant according to claim 13,wherein the 4-alpha helix bundle cytokine is IFN-τ and the valinereplaces the phenylalanine residue at position 8, 39, 68, 71, 88, 127,156, 157, 159, or 183 of SEQ ID NO.:
 12. 41. A DNA molecule encoding theprotein variant according to claim
 10. 42. A recombinant expressionvector to which the DNA molecule according to claim 41 is operablylinked.
 43. The recombinant expression vector according to claim 42,wherein the recombinant expression vector has an accession numberKCCM-10500, KCCM-10501 or KCCM-10571.
 44. A host cell transformed ortransfected with the recombinant expression vector according to claim 42or
 43. 45. A method of preparing a protein variant, comprisingcultivating the host cell according to claim 44 and isolating theprotein variant from a resulting culture.
 46. A pharmaceuticalcomposition comprising the protein variant according to claim 10 and apharmaceutically acceptable carrier.